Conditioned medium from human adult liver stem cells and its use in the treatment of liver disorders

ABSTRACT

The invention relates to cell-free compositions obtained by culturing adult-derived human liver stem/progenitor cells (ADHLSC) in cell culture medium and isolating the resulting conditioned medium (ADHLSC-CM) that has advantageous properties, such as anti-fibrotic effects. ADHLSC-CM, compositions based on ADHLSC-CM, and other related and derived products, can be used in cell culture processes or as a medicament, more particularly for the treatment of diseases involving organ injury, organ failure, in organ or cell transplantation or the pathological disruption, inflammation, degeneration, and/or proliferation of cells within a tissue or an organ, in particular within liver.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/940,897, filed Mar. 29, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/899,661, filed Dec. 18, 2015, which is a 35U.S.C. § 371 filing of International Application No. PCT/EP2014/064437,filed Jul. 7, 2014, which claims priority to European Patent ApplicationNo. 13175442.6, filed Jul. 5, 2013, each of which are incorporatedherein by reference in their entireties.

FIELD

The invention broadly pertains to medical, (bio-)pharmaceutical andpharmacological fields, and particularly concerns products, includingsubstances, compositions, and kits of parts, as well as methods and usesuseful in treating diseases and disorders, such as but withoutlimitation diseases and disorders affecting the liver.

BACKGROUND

Various conditions caused by diseased or otherwise damaged orfunctionally impaired organs may be treated by organ transplantation. Inparticular, transplantation of heart, kidneys, liver, lungs, pancreas,intestine, and thymus can routinely be performed with a reasonable rateof success. Some major drawbacks in organ transplantation, however,still remain, in particular organ shortage, the need to find acompatible donor for each recipient patient to minimise rejection of thetransplanted organ, and the need for life-long immunosuppression, assummarized in connection to liver transplantation (Zarrinpar A andBusuttil R, 2013).

In recent years, therapies based on the administration of various celltypes have been increasingly developed for regenerative medicine inhumans. Cell transplantation may provide a valuable alternative,temporary, or additional (adjunctive) therapy to organ transplantation.Mesenchymal stromal/stem cells (MSC) represent a much investigated celltype for clinical applications, such as cell-based therapies forregenerative medicine, due to the relative ease with which such cellscan be harvested from various tissues and expanded in vitro for a bettercharacterization regarding their marker profile, self-renewalproperties, and differentiation capacity (Ren G et al., 2012; Wang S etal., 2012).

Besides serving as a source of cells for replacing lost or inactiveendogenous cells, MSC also exert positive effects on tissues and organsby secreting molecules (including cytokines, extracellular matrixproteins, chemokines, growth factors, and enzymes) as soluble entitiesor microvesicles. Such mechanisms of action are behind the attributionof a variety of paracrine MSC effects on endogenous cells of majorimportance for establishing MSC-based therapies, such asimmunomodulation, tissue remodelling, cell migration, proliferation andsurvival (Atoui R and Chiu R C, 2012; Keating A. 2012; Baglio S et al.,2012).

This evidence suggests that proteins secreted by MSC (the so-called MSCsecretome) may be not only contributing to the mechanisms by which MSCexert their effects but also directly exerting some of such effectsalone, as a cell-free composition. Thus, MSC secretome has been studiedin various systems and with different techniques, showing how itscomposition and activity depend on cell origin as well as on cellculture parameters (Xiao Y et al, 2013; Lavoie J and Rosu-Myles M,2013).

MSC secretomes that have been obtained by using cells of differentorigins revealed distinct compositions and have been described inassociation to various therapeutic or in vitro uses, and to differentadministration approaches (WO 2008060788; WO 2008070868; WO 2011010966;WO 2010119176; WO 2006121445).

Relevant data have been also obtained in vivo, for example by showingthat i) the administration of intravenous bolus of conditioned medium ofbone marrow-derived MSC improves survival in a rat model of fulminanthepatic failure (Parekkadan B et al. 2007) and ii) the infusion of MSCconditioned media provides a significant survival benefit in aD-galactosamine-induced rat model of acute liver injury, preventing therelease of liver injury biomarkers (van Poll D et al., 2008). MSCconditioned media have a protective effect on hepatic cell apoptosisafter acute liver injury (Xagorari A et al., 2013), in reduced-size ratliver transplantation (Du Z et al., 2013) and in a rat model of hepaticischemia/reperfusion injuries (Pan G et al., 2012.). Besides theirinvolvement in liver regeneration and liver function recovery (WO2009057165; EP2254586; WO 2009150199), specific components of MSCsecretome can be identified as providing anti-tumour activity (CavallariC et al., 2013).

Taken together, cell/tissue regenerative therapies are clearly gainingprominence in modern medicine. However, cell-based therapies have somedrawbacks. For instance, cells may cause immune reactions whenadministered, requiring immuno-suppression; there exists some, even ifvery limited, risk that stem cells may proliferate uncontrollably whenadministered, leading to tumours or cancers, or that they maydifferentiate inappropriately in vivo; cells may lose viability and thustherapeutic efficacy if not handled carefully; etc. MSC conditionedmedia may provide some improvement, but there persists a need toidentify further and potentially improved compositions useful incell/tissue regenerative therapy, and possibly in other indications.

SUMMARY

As illustrated for representative non-limiting embodiments of theinvention in the experimental section, a cell-free composition obtainedby culturing adult-derived human liver stem/progenitor cells (ADHLSC;WO2007/071339; Najimi M et al., 2007; Khuu D et al., 2012) in cellculture medium and isolating the resulting conditioned medium(ADHLSC-CM) has unexpectedly advantageous components and properties,such as anti-fibrotic effects. ADHLSC-CM, compositions based onADHLSC-CM, and other related and derived products, can be used in cellculture processes or as a medicament, more particularly for thetreatment of diseases involving organ injury, organ failure, in organ orcell transplantation, or the pathological disruption, inflammation,degeneration, and/or proliferation of cells within a tissue or an organ,in particular within liver.

Such cell-free compositions comprise, or are derived from, cell culturemedia that have been conditioned by culturing ADHLSC, in particularthose co-expressing at least one mesenchymal marker selected from(selected from the group consisting of) CD90, CD73, CD44, vimentin andα-smooth muscle actin with at least a hepatic marker selected from(selected from the group consisting of) albumin, CD29,alpha-fetoprotein, alpha-1 antitrypsin, HNF-4 and MRP2 transporter.Still preferably ADHLSC are characterized as being albumin-positive,vimentin-positive, alpha smooth muscle actin-positive,cytokeratin-19-negative, and CD133-negative.

Conditioned medium of ADHLSC comprises useful amounts of a plurality ofspecific biological molecules, including among others growth factors,chemokines, matrix metalloproteases, and pro- and anti-inflammatorycytokines whose combination within ADHLSC-CM can be provide usefulbiological activities. The advantageous composition and properties ofADHLSC conditioned medium clearly renders this conditioned mediumqualitatively and functionally different from other conditioned media,in particular those obtained from hepatic stellate cells and mesenchymalstem cells (MSC). Consequently, ADHLSC-CM or fractions thereof providenovel products and methods that are useful in a wide variety ofapplications, in particular for treating diseases. In view of the liverorigin of ADHLSC and proven benefit of transplantation of ADHLSC intoinjured liver, such diseases may in particular but without limitationinclude diseases affecting liver.

Accordingly, an aspect of the invention provides a cell-free conditionedmedium obtainable by culturing adult-derived human liver stem/progenitorcells (ADHLSC) in a cell culture medium and separating the cell culturemedium from the cells. This cell-free conditioned medium of ADHLSC mayalso be denoted herein as “medium conditioned by ADHLSC”, or simplyADHLSC-CM. The medium may in certain preferred embodiments be obtainedby using serum-free medium.

A further aspect of the invention provides a product derived fromADHLSC-CM, which is a cell-free composition that is obtainable byfractioning ADHLSC-CM. Such fractioning may comprise applying one ormore technologies known in the art, such as for example filtering,enzymatically digesting, centrifuging, adsorbing, and/or separating bychromatography, to ADHLSC-CM.

The ADHLSC-CM, as well as the cell-free compositions that are obtainedby fractioning ADHLSC-CM, will typically contain soluble proteins and/ormicrovesicles. If ADHLSC-CM will potentially contain both these types ofcomponents, depending on the technology that may be applied forobtaining or fractioning the cell-free compositions as taught herein,this can provide an enrichment (or a selection) for both of them or foronly of one of these types of components.

In certain embodiments, the ADHLSC-CM, as well as the cell-freecompositions that are obtained by fractioning ADHLSC-CM, containssoluble proteins comprising:

-   (a) at least one of soluble proteins selected from the group    consisting of: hepatocyte growth factor (HGF), vascular endothelial    growth factor (VEGF), eotaxin (CCL11), interleukin-6 (IL-6), and    interleukin-8 (IL-8); and, optionally-   (b) at least one of soluble proteins selected from the group    consisting of matrix metalloproteases, growth factors, chemokines,    and cytokines.

Such soluble proteins may be preferably present in the ADHLSC-CM, or inthe cell-free compositions that are obtained by fractioning ADHLSC-CM,at a concentration of at least 1 ng/ml. In particular, one or more ofHGF, VEGF, CCL11, IL-6, or IL-8 (preferably all of them) may be presentat a concentration of at least 1 ng/ml.

In certain further embodiments, the ADHLSC-CM, as well as the cell-freecompositions that are obtained by fractioning ADHLSC-CM, containsmicrovesicles that are characterized and, when appropriate, selectedaccording to their size (in certain embodiments, size smaller than 0.40μm), molecular weight, and/or composition.

Particularly desired concentrations of such soluble proteins and/ormicrovesicles within ADHLSC-CM, or within the cell-free compositionsthat are obtained by fractioning ADHLSC-CM, can be obtained for exampleby appropriately concentrating (or diluting) the respective preparationat least about 5-fold, at least about 10-fold, at least about 20-fold,at least about 50-fold, or at least about 100-fold. Hence, certainembodiments provide so-concentrated or so-diluted ADHLSC-CM, as well asthe cell-free compositions that are obtained by fractioning ADHLSC-CM.

In a further embodiment, the present invention provides a method forproducing a cell-free conditioned medium comprising the steps ofculturing ADHLSC in a cell culture medium and separating the cellculture medium from ADHLSC. In certain embodiments, the method may beperformed by using a cell culture medium that is a serum-free medium, bymodifying specific conditions of cell culture, and/or by separating thecell culture medium from ADHLSC after culturing ADHLSC in the cellculture medium for at least 2 hours, at least 4 hours, at least 6 hours,at least 8 hours, at least 12 hours, or at least 24 hours. Such ADHLSCpreferably co-express at least one mesenchymal marker selected fromCD90, CD73, CD44, vimentin and α-smooth muscle actin with at least anhepatic marker selected from albumin, CD29, alpha-fetoprotein, alpha-1antitrypsin, HNF-4 and MRP2 transporter.

In a further embodiment, the present invention provides a method forproducing a cell-free composition comprising hepatocyte growth factor(HGF), vascular endothelial growth factor (VEGF), eotaxin (CCL11),interleukin-6 (IL-6), and interleukin-8 (IL-8) at a concentration of atleast 1 ng/ml comprising the steps of culturing adult-derived humanliver stem/progenitor cells (ADHLSC) in a cell culture medium andseparating the cell culture medium from the cells.

The ADHLSC-CM and cell-free compositions that are obtained byfractioning ADHLSC-CM, as intended herein, can be suitable for variousapplications. Such applications may generally encompass exposing cells,such as preferably but without limitation, cells of liver origin, invitro, ex vivo, or in vivo, to the ADHLSC-CM or the cell-freecompositions that are obtained by fractioning ADHLSC-CM.

Hence, in an aspect the invention provides the ADHLSC-CM or thecell-free compositions that are obtained by fractioning ADHLSC-CM, foruse as a medicament.

Such medical, e.g., prophylactic or therapeutic, uses may involve usingADHLSC-CM or the cell-free compositions that are obtained by fractioningADHLSC-CM alone or in combination with one or more exogenous activeingredients, which may be suitably added. Examples of such exogenousactive ingredients include cells (e.g., ADHLSC or other cells suitablefor ex vivo or in vivo applications), proteins (e.g., matrixmetalloproteases, growth factors, chemokines, cytokines, hormones,antigens, or antibodies), nutrients (e.g., sugars or vitamins) and/orchemicals (e.g., drugs with immunomodulating, anti-fibrotic, orantiviral properties) that were not initially present in ADHLSC-CM or inthe cell-free compositions, and that are known to be effective asmedicaments for the desired indication.

In a further embodiment, the present invention provides pharmaceuticalformulations comprising a pharmaceutically effective amount of ADHLSC-CMor of the cell-free compositions that are obtained by fractioningADHLSC-CM. The pharmaceutical formulations may optionally also furthercomprise a pharmaceutically effective amount of one or more exogenousactive ingredients, which may be of the type discussed above, e.g., thecells, proteins, nutrients, and/or chemicals.

Another embodiment thus provides a pharmaceutical formulation comprisinga pharmaceutically effective amount of a combination of the ADHLSC-CM orof the cell-free compositions that are obtained by fractioningADHLSC-CM, and one or more exogenous active ingredients. The exogenousactive ingredients may be of the type discussed above, e.g., the cells,proteins, nutrients, and/or chemicals.

In a further embodiment, the present invention provides ADHLSC-CM or thecell-free compositions that are obtained by fractioning ADHLSC-CM, orthe pharmaceutical formulation as defined herein, for use in thetreatment (as a prophylaxis or a therapy) of a series of disorders suchas fibrotic disorders, liver disorders, organ injury or failure, or anyother or the pathological disruption, inflammation, degeneration, and/orproliferation within an organ or a tissue. Hence, also provided is useof ADHLSC-CM or the cell-free compositions that are obtained byfractioning ADHLSC-CM, or the pharmaceutical formulation as definedherein, for the preparation of a medicament to treat a series ofdisorders such as fibrotic disorders, liver disorders, organ injury orfailure, or any other or the pathological disruption, inflammation,degeneration, and/or proliferation within an organ or a tissue.

Moreover, ADHLSC-CM or the cell-free compositions that are obtained byfractioning ADHLSC-CM, or the pharmaceutical formulation as definedherein, or a combination of two or more thereof, can be used in organ orcell transplantation, in a preferred example, as a subsidiary treatmentthat can be administered before, after, or when performingtransplantation. Also provided is thus the use of ADHLSC-CM or thecell-free compositions that are obtained by fractioning ADHLSC-CM, orthe pharmaceutical formulation as defined herein, or a combination oftwo or more thereof, for the preparation of a medicament for organ orcell transplantation, in a preferred example, as a subsidiary treatmentthat can be administered before, after, or when performingtransplantation.

In a further aspect, the present invention provides a method of treatinga disorder (such as in particular a fibrotic disorder, liver disorder,or organ injury or failure) in a subject in need of said treatmentcomprising the administration of a therapeutically or prophylacticallyeffective amount of ADHLSC-CM, or the cell-free composition that isobtainable by fractioning ADHLSC-CM, or the pharmaceutical formulationas defined above, to the subject, applying the appropriate method andfrequency of administration.

These and further aspects and preferred embodiments of the invention aredescribed in the following sections and in the appended claims. Thesubject-matter of the appended claims is hereby specificallyincorporated in this specification.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C: secretome profile in the conditioned culture medium ofadult-derived human liver stem/progenitor cells (ADHLSC-CM) and hepaticstellate cells (HSC-CM) in absence of serum. HGF secretion issignificantly higher in ADHLSC-CM (FIG. 1A) but a broader analysis ofgrowth factors and cytokines secretion in ADHLSC-CM and HSC-CM showsmany other proteins are more secreted in ADHLSC-CM (FIGS. 1B-1C). Forthe analysis of statistically relevant differences between ADHLSC-CM andHSC-CM, *** denotes a p value <0.001, ** denotes a p value <0.01, and *denotes a p value <0.05. ns: not significant.

FIGS. 2A-2B: effect of ADHLSC on HSC number and viability in theTranswell® co-culture system. The effect of exposing HSC to ADHLSC isdetermined by comparing HSC number when cultured in absence of ADHLSC(control, 0/1) or when cultured at low ADHLSC/HSC ratio (1/100) atdistinct time points (FIG. 2A), and then using CCK-8 biochemical assayfor determining post-seeding HSC number (FIG. 2B). For the analysis ofstatistically relevant differences between the two conditions, **denotes a p value <0.01. n: number of donors for which the experimentwas performed.

FIGS. 3A-3B: effect of ADHLSC on HSC proliferation and adherence. Theassay is based on the determination of floating or adherent HSC in theTranswell® co-culture system after culturing HSC for 24 hours inpresence of ADHLSC that secrete biological molecules passing across thecollagen-treated membrane separating the two chambers and having a poresize of 0.4 μm (FIG. 3A) or by directly culturing HSC in ADHLSC-CM orHSC-CM (FIG. 3B). The effect on the number of floating or adherent HSChas been also tested at lower ADHLSC/HSC ratios (1/1000 and 1/10000),but the number of floating cells also decreased consequently, suggestingthat 1/100 ratio is a particularly effective experimental condition forevaluating the effect of ADHLSC. For the analysis of statisticallyrelevant differences between the two conditions, ** denotes a p value<0.01 and* denotes a p value <0.05. n: number of donors for which theexperiment was performed.

FIGS. 4A-4B: analysis of ADHLSC-CM and HSC-CM effects on HSC adherence.Post-seeding floating HSC (FIG. 4A) and adherent (FIG. 4B) HSC aredetermined at intermediate time points after adding ADHLSC-CM or HSC-CM.For the analysis of statistically relevant differences between theeffects of ADHLSC-CM and HSC-CM, ** denotes a p value <0.01 and *denotes a p value <0.05. n: number of donors for which the experimentwas performed.

FIGS. 5A-5C: effects of ADHLSC and ADHLSC-CM on HSC cell cycle andproliferation. The effect is assessed by Propidium Iodide staining(FIGS. 5A-5B) and by Ki-67 immunostaining (FIG. 5C), showing thequantitative effect of exposing HSC to ADHLSC in the Transwell®co-culture system (FIG. 5A) or by adding directly ADHLSC-CM (FIGS.5B-5C) on the percentage of HSC in a given state. The Ki67immunostaining of HSC showed a significant decrease of the number ofimmunostained nuclei of HSC pre-incubated for 24 hours with ADHLSC-CM.Similarly to the effect on floating or adherent HSC (see FIG. 3), atlower ADHLSC/HSC ratios (1/1000 and 1/10000), the effect on the numberof HSC that are in either G0/G1 Phase or G2/M phase is not statisticallysignificant, confirming that 1/100 ratio, as shown in the Figure, is aparticularly effective experimental condition for evaluating the effectof ADHLSC secretome. For the analysis of statistically relevantdifferences between the different conditions or between ADHLSC-CM andHSC-CM, *** denotes a p value <0.001 and * denotes a p value <0.05;other relevantp values are indicated. ns: not significant. n: number ofdonors for which the experiment was performed.

FIGS. 6A-6B: effect of ADHLSC on the HSC secretion of factors relevantto fibrogenesis in the Transwell® co-culture system, such as collagentype I (FIG. 6A) and HGF or IL-6 (FIG. 6B). After being exposed toADHLSC for 24 hours, the medium in chamber containing HSC wassubstituted with serum-free medium and incubated further 24 hours beforemeasuring the concentration of these proteins that are secreted in thisserum-free medium. For the analysis of statistically relevantdifferences between the effects of different conditions, ** denotes a pvalue <0.01. n: number of donors for which the experiment was performed.

DESCRIPTION OF EMBODIMENTS

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. The terms also encompass“consisting of” and “consisting essentially of”, which enjoywell-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, preferably +1-5%or less, more preferably +/−1% or less, and still more preferably+/−0.1% or less of and from the specified value, insofar such variationsare appropriate to perform in the disclosed invention. It is to beunderstood that the value to which the modifier “about” refers is itselfalso specifically, and preferably, disclosed.

Whereas the terms “one or more” or “at least one”, such as one or moremembers or at least one member of a group of members, is clear per se,by means of further exemplification, the term encompasses inter alia areference to any one of said members, or to any two or more of saidmembers, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members,and up to all said members. In another example, “one or more” or “atleast one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included toexplain the context of the invention. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge in any country as of the prioritydate of any of the claims.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Alldocuments cited in the present specification are hereby incorporated byreference in their entirety. In particular, the teachings or sections ofsuch documents herein specifically referred to are incorporated byreference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the invention. When specific terms aredefined in connection with a particular aspect of the invention or aparticular embodiment of the invention, such connotation is meant toapply throughout this specification, i.e., also in the context of otheraspects or embodiments of the invention, unless otherwise defined.

In the following passages, different aspects or embodiments of theinvention are defined in more detail. Each aspect or embodiment sodefined may be combined with any other aspect(s) or embodiment(s) unlessclearly indicated to the contrary. In particular, any feature indicatedas being preferred or advantageous may be combined with any otherfeature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to a person skilled in the art from this disclosure, in one ormore embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments,combinations of features of different embodiments are meant to be withinthe scope of the invention, and form different embodiments, as would beunderstood by those in the art. For example, in the appended claims, anyof the claimed embodiments can be used in any combination.

As illustrated for representative non-limiting embodiments of theinvention in the experimental section, the present inventors realisedthat a cell-free composition obtained by culturing adult-derived humanliver stem/progenitor cells (ADHLSC; WO2007/071339; Najimi M et al.,2007; Khuu D et al., 2012) in cell culture medium and isolating theresulting conditioned medium (ADHLSC-CM) has unexpectedly advantageouscomponents and properties, such as anti-fibrotic effects. ADHLSC-CM,compositions based on ADHLSC-CM, and other related and derived products,can be used in cell culture processes or as a medicament, moreparticularly for the treatment of diseases involving organ injury, organfailure, or the pathological disruption, inflammation, degeneration,and/or proliferation of cells within a tissue or an organ, in particularwithin liver.

The term “adult-derived human liver stem or progenitor cells”,abbreviated as “ADHLSC”, as used herein, specifically denotes the humanprogenitor or stem cell originated from human adult liver as disclosedin WO 2007/071339 and described in the relevant literature (Najimi etal. 2007; Khuu D et al., 2012), all incorporated by reference herein.Hence, the conditioned medium of ADHLSC as taught herein (ADHLSC-CM) isobtained by culturing ADHLSC.

Suitably, ADHLSC are obtained by using samples of adult liver, and arecharacterised by co-expression of at least one mesenchymal marker(preferably one, more than one or all of the markers CD90, CD73, CD44,vimentin and α-smooth muscle actin) with hepatic markers (preferably oneor more of albumin, CD29, alpha-fetoprotein, alpha-1 antitrypsin, HNF-4and MRP2 transporter). In particular ADHLSC typically co-expressα-smooth muscle actin and/or vimentin and albumin (ALB), but are alsocharacterized by other criteria such as the morphology (mesenchymal-likemorphology with flattened form broad cytoplasm, growing in monolayers),the capability of differentiating into hepatocytes or hepatocyte-likecells and not differentiating into mesodermal cell types, and the lackof expression of other markers such cytokeratin-7, cytokeratin-19,Oct-4, CD45, CD34, CD133, and CD117. Thus, exemplary ADHLSC that can beused for obtaining ADHLSC-CM are albumin-positive, vimentin-positive,alpha smooth muscle actin-positive, cytokeratin-19-negative, andCD133-negative.

By means of further guidance, ADHLSC may be characterised in that itco-expresses at least one mesenchymal marker with the hepatocyte markeralbumin (ALB). More particularly, ADHLSC may co-express α-smooth muscleactin (ASMA) and ALB. Even more particularly, ADHLSC may co-expressvimentin, ASMA and ALB. Still more particularly, ADHLSC may co-expressASMA and ALB, or vimentin, ASMA and ALB, and be negative forcytokeratin-19 (CK-19). Negativity of ADHLSC for CK-19 may beparticularly determined at protein level, e.g., by immunocytochemistry.Yet more particularly, ADHLSC may express CD90, CD73, CD44, vimentin,ASMA and ALB, and be negative for CK-19. ADHLSC typically also expressesfurther markers, such as CD29, CD13, cytochrome P450 3A4 (CYP3A4),CYP1B1, alpha fetoprotein (AFP), and alpha-anti-trypsin. In a particularexample, ADHLSC may thus be characterised as CD90, CD29 and CD44positive, albumin-positive, vimentin-positive and ASMA-positive, andnegative for CD45, CD34, CD117 and CK-19. In another example, ADHLSC maybe characterised as expressing CD90, CD73, CD29, CD44, CD13, vimentin,ASMA, ALB, AFP, CYP3A4 and alpha-anti-trypsin. In a further example,ADHLSC may be characterised as expressing CD90, CD73, CD29, CD44, CD13,vimentin, ASMA, ALB, AFP, CYP3A4 and alpha-anti-trypsin, and negativefor CK-19 and CK-7. In a yet further example, ADHLSC may becharacterised as expressing CD90, CD73, CD29, CD44, CD13, vimentin,ASMA, ALB, AFP, CYP3A4 and alpha-anti-trypsin, and negative for CK-19,CK-7, CD133, CD117, CD45, CD34 and HLA-DR. Positivity and negativity ofADHLSC for the various markers may be preferably determined at proteinlevel, e.g., by immunocytochemistry. By means of example and withoutlimitation, ADHLSC may display marker expression profile as found in thecells as deposited by Université catholique de Louvain (represented byProfessor Bernard Coulie, Rector of UCL from 2004 to 2009) on Feb. 20,2006 under the Budapest Treaty with the Belgian Coordinated Collectionsof Microorganisms (BCCM/LMBP) under accession number LMBP 6452CB. Itshall be appreciated that cell lines derived of ADHLSC cells are alsoencompassed in the term.

Wherein a cell is said to be positive for (or to express, i.e., compriseexpression of) a particular marker, this means that a skilled personwill conclude the presence or evidence of a distinct signal, e.g.,antibody-detectable (e.g., immunocytochemistry or immunoblotting) forthat marker when carrying out the appropriate measurement, compared tosuitable (negative) controls. Where the method allows for quantitativeassessment of the marker, positive cells may on average generate asignal that is significantly different from the control, e.g., butwithout limitation, at least 1.5-fold higher than such signal generatedby control cells, e.g., at least 4-fold, at least 5-fold, or at least10-fold higher, or (preferably) even higher.

The expression of the above cell-specific markers can be detected usingany suitable immunological technique known in the art (such asimmuno-cytochemistry or affinity adsorption, Western blot analysis,FACS, ELISA, protein microarrays, etc.) or any suitable sequencingtechnologies for identifying and quantifying proteins from biologicalsamples. Sequence data for markers listed in this disclosure are knownand can be obtained from public databases such as GenBank(http://www.ncbi.nlm.nih.gov/) or Uniprot (http://www.uniprot.org).

By means of further guidance, the terms “progenitor” or “progenitorcell” are synonymous and generally refer to an unspecialised orrelatively less specialised and proliferation-competent cell which canunder appropriate conditions give rise to at least one relatively morespecialised cell type, such as inter alia to relatively more specialisedprogenitor cells or eventually to terminally differentiated cells. Aprogenitor cell may “give rise” to another, relatively more specialisedcell when, for example, the progenitor cell differentiates to becomesaid other cell without previously undergoing cell division, or if saidother cell is produced after one or more rounds of cell division and/ordifferentiation of the progenitor cell.

The term “stem cell” generally refers to a progenitor cell capable ofself-renewal, i.e., which can under appropriate conditions proliferatewithout differentiation. The term encompasses stem cells capable ofsubstantially unlimited self-renewal, i.e., wherein at least a portionof the stem cell's progeny substantially retains the unspecialised orrelatively less specialised phenotype, the differentiation potential,and the proliferation capacity of the mother stem cell; as well as stemcells which display limited self-renewal, i.e., wherein the capacity ofthe stem cell's progeny for further proliferation and/or differentiationis demonstrably reduced compared to the mother cell.

The term “adult stem or progenitor cell” as used herein refers to a stemor progenitor cell present in or obtained from (such as isolated from)an organism at the foetal stage or after birth, such as for example atany one of the stages commonly referred to as “newborn”, “infant”,“child”, “youth”, “adolescent” or “adult”. For example, ADHLSC may beoriginated from human foetuses, or from human subjects at any time afterbirth, preferably full term, e.g., 0-1 month of age after birth, or atleast 1 month of age after birth, e.g., at least 2 months, at least 3months, e.g., at least 4 months, at least 5 months, e.g., at least 6months age after birth, such as, for example, 1 year or more, 5 years ormore, at least 10 years or more, 15 years or more, 20 years or more, or25 years or more of age after birth.

ADHLSC can be suitably isolated from human liver by the methods taughtin WO 2007/071339 and further described by Najimi et al. 2007, bothincorporated by reference herein.

ADHLSC can be also suitably genetically modified before being used forproducing ADHLSC-CM by the methods taught in WO 2007/071339, inparticular for increasing the replicative capacity of ADHLSC, forenhancing ADHLSC growth and/or activity, for constitutively or induciblyover-expressing a polypeptide normally expressed (and secreted, or not)by hepatocytes, an antibody, or a hormone.

Hence, ADHLSC is obtainable or directly obtained by a method comprising:(a) disassociating adult liver or a part thereof to obtain a populationof primary cells from the said adult liver or part thereof, (b) platingthe primary cell population onto a substrate which allows adherence ofcells thereto, and (c) culturing cells from the primary cell population,which have adhered to the said substrate, for at least 7 days,preferably at least 10, at least 13, or at least 15 days until thecultured cell population is at least 40% and preferably at least 70%confluent, thereby allowing for the emergence and proliferation of thestem/progenitor cells in the cell population; and (d) increasing theproportion of the progenitor or stem cells in the cell population bypassaging the cell population at least once and preferably at least twotimes, thereby preferably obtaining a substantially homogeneouspopulation of the stem/progenitor cells. In step (b) preferably theliver parenchymal cell population is plated. Typically, the cellpopulation may be passaged between 2 and 8 times in step (d), duringwhich ADHLSC are expanded and produce ADHLSC-CM that can be isolated andcharacterized.

In a particular example, the method may comprise: (a) disassociatinghuman adult liver or a part thereof to form a population of primarycells from the said adult liver or part thereof, (b) plating the primarycell population onto a substrate which allows adherence of cellsthereto, (c) culturing cells from the primary cell population, whichhave adhered to the said substrate, for at least 7 days in a culturemedium comprising serum and a combination of at least two exogenouslyadded growth factors chosen from epidermal growth factor (EGF),dexamethasone, and insulin, preferably at least dexamethasone andinsulin, more preferably dexamethasone and insulin but not EGF, and (d)exchanging the medium for basal medium comprising high glucose at aconcentration between 3 000 mg/L and 6 000 mg/L and further culturingthe cells, whereby ADHLSC emerges and proliferates. In step (b)preferably the liver parenchymal cell population is plated. Emergence ofADHLSC may be facilitated by allowing the cells to become about 70%confluent and passaging the cells at least once, and typically between 2and 8 times.

In another example, the method may comprise: (a) disassociating,preferably by two-step collagenase method, adult liver or a part thereoffrom a human subject, to form a population of primary cells from thesaid adult liver or part thereof; (b) plating the primary cellpopulation onto collagen type I coated substrate in Williams Medium Ecomprising foetal calf serum, preferably 10% (v/v), EGF, preferably 25ng/ml, insulin, preferably 10 pg/ml, and dexamethasone, preferably 1 μM;(c) allowing adherence of cells from the primary cell population to thesaid substrate for 24 hours and thereafter exchanging the medium forfresh medium having composition as in (b); (d) culturing the cells inthe said medium of (c) during two weeks, preferably 15 days; (e)exchanging the medium for DMEM comprising high glucose and FCS,preferably 10%, and further culturing the cells, whereby the progenitoror stem cells of the invention emerge and proliferate; (f) allowing thecells to become about 70% confluent and passaging the cells at leastonce and preferably at least two times, wherein the cells are platedonto the substrate as in (b) and cultured in a medium as in (e). In step(b) preferably the liver parenchymal cell population is plated.Typically, the cell population may be passaged between 2 and 8 times instep (f).

It shall be appreciated that in the above methods, more specificallyduring culturing and passaging the cells, the emerging ADHLSC cellsbecome gradually overrepresented in the cell population, until asubstantially homogeneous population of ADHLSC results. This processthus does not entail selection of individual colonies of cells in thesesteps.

The specific example as described by Najimi et al. 2007 (supra) is asfollows. Single cell suspensions were resuspended in Williams' E medium(Invitrogen) supplemented with 10% foetal calf serum (FCS) (Perbio,Hyclone), 25 ng/ml EGF (Peprotech), 10 μg/ml insulin, 1 μMdexamethasone, and 1% penicillin/streptomycin (P/S) (Invitrogen). Thecells were plated on six-well rat tail collagen I-coated plates (GreinerBio-one) and cultured at 37° C. in a fully humidified atmospherecontaining 5% CO2. After 24 h, medium was changed in order to eliminatethe non-adherent cells and thereafter renewed every 3 days whereas theculture was microscopically followed every day. Culture medium was thenswitched to DMEM with high glucose concentrations (Invitrogen)supplemented with 10% FCS and 1% P/S in order to accelerate theelimination of hepatocytes. A cell type with mesenchymal-like morphologythen spontaneously emerged, proliferated, and filled the empty space inthe well plate as observed by phase contrast microscopy. When reaching70% confluence, cells were lifted with 0.25% trypsin and 1 mM EDTA andre-plated at a density of 1×10⁴ cells/cm². ADHLSC cells from third toeight passage were characterised further.

Hence, in certain embodiments, ADHLSC may be obtainable by or directlyobtained by any of the above methods.

Accordingly, a method for producing a cell-free conditioned medium astaught herein may comprise the step of obtaining ADHLSC by any of theabove methods, culturing ADHLSC in a cell culture medium, and separatingthe cell culture medium from ADHLSC.

The synonymous phrases “cell-free” and “free of cells” are generallywell-understood, and in the present context may particularly signifythat a composition such as a conditioned medium essentially does notcontain (ADHLSC) cells, especially essentially does not contain viable(ADHLSC) cells. The degree to which a composition such as a conditionedmedium is free of cells tends to be largely determined by theeffectiveness of available methods for separating cells from culturemedia, such as, for example, centrifugation or filtration, orrepetitions and/or combinations of such methods. For practical purposes,a composition such as a conditioned medium may be considered cell-freewhen it contains 5×10² or fewer cells/ml, preferably 100 or fewercells/ml, more preferably 50 or fewer cells/ml, even more preferably 25or fewer cells/ml, yet more preferably 10 or fewer cells/ml, still morepreferably 10 or fewer cells/ml or 5 or fewer cells/ml, and mostpreferably no (i.e., 0) cells/ml; preferably these counts denote viablecells. Conventional cell counting methods may be used, such as lightmicroscopy, or flow cytometry, or plating and colony forming units (CFU)determination. Conventional cell viability determination methods may beused, such as dye (e.g., trypan blue or propidium iodide) exclusionassays.

The term “medium” as used herein broadly encompasses any cell culturemedium conducive to maintenance and/or proliferation of cells; moreparticularly conducive to maintenance of ADHLSC, preferably conducive toproliferation of ADHLSC. Typically, the medium will be a liquid culturemedium, which facilitates easy manipulation (e.g., decantation,pipetting, centrifugation, filtration, and such) thereof.

Suitable culture medium is defined in the specific references(WO2007/071339; Khuu D et al., 2012) and in the Examples but it can beadapted for obtaining the enrichment (or the depletion) of specificelements. For example, ADHLSC-CM can be obtained by using serum-freemedium and/or in presence or absence of specific nutrients. The densityand the number of ADHLSC in the cell culture can be adapted to thedesired volume and/or protein concentration of the ADHLSC-CM.

Typically, the medium will comprise a basal medium formulation as knownin the art. Many basal media formulations (available, e.g., from theAmerican Type Culture Collection, ATCC; or from Invitrogen, Carlsbad,Calif.) can be used to culture ADHLSC cells herein, including but notlimited to Eagle's Minimum Essential Medium (MEM), Dulbecco's ModifiedEagle's Medium (DMEM), alpha modified Minimum Essential Medium(alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco'sMedium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Leibovitz L-15,DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium199, Waymouth's MB 752/1 or Williams Medium E, and modifications and/orcombinations thereof. Compositions of the above basal media aregenerally known in the art and it is within the skill of one in the artto modify or modulate concentrations of media and/or media supplementsas necessary for the cells cultured. A particularly preferred basalmedium, especially for culturing ADHLSC, may be DMEM.

Such basal media formulations contain ingredients necessary formammalian cell maintenance and proliferation, which are known per se. Bymeans of illustration and not limitation, these ingredients may includeinorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P andpossibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES,bicarbonate), nucleotides, nucleosides and/or nucleic acid bases,ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g.,glutathione) and sources of carbon (e.g., glucose, sodium pyruvate,sodium acetate), etc.

By means of example, a medium for culturing mammalian cells such asADHLSC may contain between 1.0 g/L and 10.0 g/L D-glucose, preferablybetween 3.0 g/L and 6.0 g/L D-glucose, more preferably between 4.0 g/Land 5.0 g/L D-glucose, and most preferably 4.50 g/L glucose.

For use in culture, basal media can be supplied with one or more furthercomponents. For example, additional supplements can be used to supplythe cells with the necessary trace elements and substances for optimalADHLSC maintenance, growth, and/or expansion. Furthermore, antioxidantsupplements may be added at appropriate concentrations, e.g.,β-mercaptoethanol or N-acetyl-L-cysteine. While many basal media alreadycontain amino acids, some amino acids may be supplemented later, e.g.,L-glutamine, which is known to be less stable when in solution. A mediummay be further supplied with antibiotic and/or antimycotic compounds,such as, typically, mixtures of penicillin and streptomycin, and/orother compounds, exemplified but not limited to, amphotericin,ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin,mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin,polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin,and zeocin.

Lipids and lipid carriers can also be used to supplement cell culturemedia. Such lipids and carriers can include, but are not limited tocyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleicacid and oleic acid conjugated to albumin, unconjugated linoleic acid,linoleic-oleic-arachidonic acid conjugated to albumin, oleic acidunconjugated and conjugated to albumin, among others. Albumin cansimilarly be used in fatty-acid free formulations.

Also contemplated is supplementation of cell culture media withmammalian plasma or sera.

Plasma or sera often contain cellular factors and components thatfacilitate cell viability and expansion. Optionally, plasma or serum maybe heat inactivated. Heat inactivation is used in the art mainly toremove the complement. Heat inactivation typically involves incubatingthe plasma or serum at 56° C. for 30 to 60 min, e.g., 30 min, withsteady mixing, after which the plasma or serum is allowed to graduallycool to ambient temperature. A skilled person will be aware of anycommon modifications and requirements of the above procedure.Optionally, plasma or serum may be sterilised prior to storage or use.Usual means of sterilisation may involve, e.g., filtration through oneor more filters with pore size smaller than 1 μm, preferably smallerthan 0.5 μm, e.g., smaller than 0.45 μm, 0.40 μm, 0.35 μm, 0.30 μm or0.25 μm, more preferably 0.2 μm or smaller, e.g., 0.15 μm or smaller,0.10 μm or smaller. Suitable sera or plasmas for use in media as taughtherein may include human serum or plasma, or serum or plasma fromnon-human animals, preferably non-human mammals, such as, e.g.,non-human primates (e.g., lemurs, monkeys, apes), foetal or adultbovine, horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum orplasma, etc, or any combination of such. In certain preferredembodiments, a medium as taught herein may comprise bovine serum orplasma, preferably foetal bovine (calf) serum or plasma, more preferablyfoetal bovine (calf) serum (FCS or FBS). In other preferred embodiments,media as taught herein may comprise human serum or plasma, such asautologous or allogeneic human serum or plasma, preferably human serum,such as autologous or allogeneic human serum, more preferably autologoushuman serum or plasma, even more preferably autologous human serum.

In certain preferred embodiments, serum or plasma can be substituted inmedia as taught herein by serum replacements, such as to provide forserum-free media (i.e., chemically defined media). The provision ofserum-free media may be advantageous particularly with view toadministration of the media or fraction(s) thereof to subjects,especially to human subjects (e.g., improved bio-safety). By the term“serum replacement” it is broadly meant any a composition that may beused to replace the functions (e.g., cell maintenance and growthsupportive function) of animal serum in a cell culture medium. Aconventional serum replacement may typically comprise vitamins, albumin,lipids, amino acids, transferrin, antioxidants, insulin and traceelements. Many commercialized serum replacement additives, such asKnockOut Serum Replacement (KOSR), N2, B27, Insulin-Transferrin-SeleniumSupplement (ITS), and G5 are well known and are readily available tothose skilled in the art.

Plasma or serum or serum replacement may be comprised in media as taughtherein at a proportion (volume of plasma or serum or serumreplacement/volume of medium) between about 0.5% v/v and about 40.0%v/v, preferably between about 5.0% v/v and about 20.0% v/v, e.g.,between about 5.0% v/v and about 15.0% v/v, more preferably betweenabout 8.0% v/v and about 12.0% v/v, e.g., about 10.0% v/v.

A particularly preferred medium, especially for culturing ADHLSC, may beDMEM containing between 3.0 g/L and 6.0 g/L D-glucose, more preferablybetween 4.0 g/L and 5.0 g/L D-glucose, most preferably 4.50 g/L glucose;supplemented with between 5.0% v/v and 15.0% v/v foetal calf serum(FCS), preferably between 8.0% v/v and 12.0% v/v FCS, most preferably10.0% v/v FCS; and suitably supplemented with antibiotics, e.g.,penicillin and streptomycin. FCS is preferably substituted by a suitableamount of serum-replacement.

The term “conditioned medium” refers to a medium that has been exposedto (i.e., contacted with, cultured with) cells grown in culture for atime sufficient to include at least one additional component in themedium, said component produced by the cells, that was not present inthe medium before exposing the same to the cells. In other words, a“conditioned medium” may be deemed as a composition comprising cellsecretion products, such as inter alia cell secretion proteins andcellular metabolites, which has previously supported the maintenanceand/or the proliferation of cells.

The period of time sufficient to include said at least one additionalcomponent in the medium may in particular be sufficient to achievesecretion by ADHLSC of mixed secretion products (including secretedproteins and microvesicles) into the medium. By means of example, saidperiod of time may be at least about 1 hour, preferably at least about 3hours, more preferably at least about 6 hours, even more preferably atleast about 12 hours, still more preferably at least about 18 hours, andyet more preferably at least about 24 hours, such as for example atleast about 36 hours or at least about 48 hours. Typically, said periodof time will be no more than about 72 hours, more typically no more thanabout 60 hours, even more typically no more than about 48 hours.

A conditioned medium such as ADHLSC-CM may thus be obtainable by ordirectly obtained by culturing ADHLSC in a cell culture medium, therebyconditioning the medium, and separating the cell culture medium from thecells, thereby obtaining the conditioned medium. The terms “culturing”or “cell culture” are common in the art and broadly refer to maintenanceof cells and potentially proliferation of cells in vitro. Typically,animal cells, such as mammalian cells, such as human cells, are culturedby exposing them to (i.e., contacting them with) a suitable cell culturemedium in a vessel or container adequate for the purpose of anexperiment (e.g., a 96-, 24-, or 6-well plate, a T-25, T-75, T-150 orT-225 flask, or a cell factory), at art-known conditions conducive to invitro cell culture, such as temperature of 37° C., 5% v/v CO₂ and >95%humidity.

Regarding cell culture conditions, the material for cell culture (e.g.,plates, flasks, or bioreactors) can be selected among those defined inWO2007/071339, or made available more recently and identified as adaptedfor growing cells like MSC or other primary cells in particular usingconditions that make possible to generate ADHLSC-CM according to GoodManufacturing Practice requirements for cell-based pharmaceuticalproducts. Preferably, any appropriate coating, treatment or other methodfor preparing the surface where primary cells or MSC (and ADHLSC inparticular) adhere and proliferate can be used. For example, the surfacecan be coated with biological materials such as extracellular matrixattachment and adhesion proteins (e.g. collagen, laminin, fibronectin,heparin sulfate, hyaluronidate, or chondroitin sulfate, either appliedindividually or as mixtures), matrices (e. g. Matrigel™; BDBiosciences). Otherwise, pre-treated surface (such as CELLBIND™ cellculture materials, Ultra-Web® Synthetic Surfaces, or surfaces treatedwith synthetic peptides) can be used. Other cell culture conditions thatcan be finely adapted for obtaining ADHLSC-CM having the desiredcomposition and properties include temperature, cell density at seeding,and oxygen tension.

The term “in vitro” generally denotes outside, or external to, animal orhuman body. The term “ex vivo” typically refers to tissues or cellsremoved from an animal or human body and maintained or propagatedoutside the body, e.g., in a culture vessel. The term “in vitro” as usedherein should be understood to include “ex vivo”. The term “in vivo”generally denotes inside, on, or internal to, animal or human body.

When preparing a conditioned medium, cells by the action of which themedium is to be conditioned may be contacted with the medium at varietyof initial cell densities. By means of example, adherent cells, i.e.,cells capable of adhering to tissue culture plastic or to suitableadherent substrate(s), such as ADHLSC, may be contacted with the mediumat initial cell confluence of at least about 50%, e.g., at least about55%, or of at least about 60%, e.g., at least about 65%, or of at leastabout 70%, e.g., at least about 75%, or of at least about 80%, e.g., atleast about 85%, or of at least about 90%, e.g., at least about 95%,such as 96%, 97%, 98%, 99%, or even 100% initial cell confluence. Theterm “confluence” refers to density of cultured cells in which the cellscontact one another covering substantially all of the surfaces availablefor cell proliferation (i.e., fully confluent).

By means of example, adherent cells, such as ADHLSC, may be contactedwith the medium at initial cell density of at least about 1000 cells/cm²growth surface area, e.g., of at least about 10 000 cells/cm², or of atleast about 25 000 cells/cm², or of at least about 50000 cells/cm²growth surface area, e.g., at least about 75 000 cells/cm², or of atleast about 100 000 cells/cm², e.g., at least about 125 000 cells/cm²initial cell density. For example, adherent cells, such as ADHLSC, maybe contacted with the medium at initial cell density of no more thanabout 200 000 cells/cm², typically no more than about 175 000 cells/cm²,more typically no more than about 150 000 cells/cm² initial celldensity. Hence, by means of example, adherent cells, such as ADHLSC, mayusually be contacted with the medium at initial cell density of betweenabout 50 000 cells/cm² and about 125 000 cells/cm², such as, e.g.,between about 75 000 cells/cm² and about 100 000 cells/cm², such as,e.g., about 80 000 cells/cm².

When preparing a conditioned medium, the medium to be conditioned may beprovided at volumes commonplace in tissue culture. Typically, adherentcells, such as ADHLSC, may be contacted with between about 0.10 mL/cm²growth surface area and about 0.40 mL/cm² growth surface area of medium,more typically between about 0.15 mL/cm² and about 0.35 mL/cm², evenmore typically between about 0.20 mL/cm² and about 0.30 mL/cm².

In certain embodiments, particularly but without limitation when theconditioned medium or product(s) derived there from are intended foradministration to subjects, especially to human subjects, the medium maylack xenogeneic serum or plasma, i.e., serum or plasma originating froman organism of a species distinct from the species of the subject towhich the conditioned medium or product(s) derived there from are to beadministered. In an example, any plasma or serum contained in the mediummay only be allogeneic serum or plasma, i.e., serum or plasmaoriginating from a member of the same species as the species of thesubject to which the conditioned medium or product(s) derived there fromare to be administered, but not from the subject. In another example,any plasma or serum contained in the medium may only be autologous serumor plasma, i.e., serum or plasma originating from the subject to whichthe conditioned medium or product(s) derived there from are to beadministered. In yet another, particularly preferred example, the mediummay lack any serum or plasma, i.e., serum-free medium. The provision ofmedia of these embodiments can improve the bio-safety and/orimmunological profile of the medium.

The method for producing a cell-free conditioned medium may thuscomprise the steps of culturing ADHLSC, that in particular co-express atleast one mesenchymal marker selected from CD90, CD73, CD44, vimentinand α-smooth muscle actin with at least an hepatic marker selected fromalbumin, CD29, alpha-fetoprotein, alpha-1 antitrypsin, HNF-4 and MRP2transporter, in a cell culture medium and separating the cell culturemedium from ADHLSC. This method can be performed by using a cell culturemedium that is a serum-free medium, by modifying specific conditions ofcell culture, and/or by separating the cell culture medium from ADHLSCafter culturing ADHLSC at given time points (e.g. at least 2 hours, atleast 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, orat least 24 hours) at the scope of obtaining ADHLSC-CM having acomposition enriched (or depleted) in soluble proteins and/ormicrovesicles that are either degradated (or unstable) within theconditioned media or secreted by ADHLSC not in regular manner but onlybefore or after a certain number of hours (and thus not progressivelyaccumulated in the ADHLSC-CM). Relevant time points can be very short(e.g. 2 hours or less) or longer such as at 24 hours (as in theExamples), at 36 hours or more hours. By obtaining samples of ADHLSC-CMat these time points or at intermediate ones (such as 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, or 18 hours) and testing such samplesfor their composition and/or activities, the optimal timing forobtaining the desired ADHLSC-CM can be determined.

In certain further embodiments, particularly but without limitation whenthe conditioned medium or product(s) derived there from are intended foradministration to subjects, especially to human subjects, the medium maybe manufactured such as to ensure compliance with good manufacturingpractice (GMP) guidelines. In certain embodiments, the conditionedmedium may be sterilised, such as conveniently by passing through amicrobiological filter having pore size about 0.20 μm, e.g., between0.20 μm and 0.25 μm, e.g., 0.21 μm, 0.22 μm, 0.23 μm, or 0.24 μm.

A conditioned medium may be separated from cells used to condition themedium by any available technique. Conventional techniques include forexample removal of the medium from a culture vessel by decantation orpipetting, centrifugation of the medium to pellet cells, cell fragmentsand particulates present therein (e.g., centrifugation at between about100×g and about 2 000×g, such as between about 500×g and about 1 500×g,such as at about 1 000×g, for between about 5 min and about 30 min, suchas between about 10 min and 20 min, such as for about 15 min),filtration of the medium to filter away cells, cell fragments andparticulates present therein (e.g., filtration through a standardmicrobiological filter having pore size about 1.0 μm or less, preferablyabout 0.8 μm or less, more preferably about 0.6 μm or less, yet morepreferably about 0.4 μm or less, such as preferably about 0.2 μm). Itshall be appreciated that repetitions and/or combinations of suchmethods may be employed to attain comparatively more completeseparation.

Hence, the separation of ADHLSC-CM from ADHLSC can be performed bysimply transferring the supernatant of the ADHLSC culture container (bydecantation or pipetting) into a separate container and, optionally,repeating and/or combining filtration (e.g., filtration through astandard microbiological filter having pore size about 1.0 μm or less,preferably about 0.8 μm or less, more preferably about 0.6 μm or less,yet more preferably about 0.4 μm or less, such as preferably about 0.2μm) or shortly centrifugation this cell culture supernatant at low speed(e.g., centrifugation at between about 100×g and about 2 000×g, such asbetween about 500×g and about 1 500×g, such as at about 1 000×g, forbetween about 5 min and about 30 min, such as between about 10 min and20 min, such as for about 15 min), at the scope of pelleting anyremaining cell, cell debris, or particulate. In this manner, a cell-freepreparation is obtained as supernatant of the centrifugation and it canbe then used for determining identity and concentration of thebiological molecules that are present, such as soluble proteins ormicrovesicles as defined below, and according to commonly availabletechnology such as immunoassays, spectrometry, or enzymatic assays.

As set forth in the experimental section, the inventors demonstratedthat in a co-culture system where ADHLSC were separated from HepaticStellate Cells (HSC) by a 0.4 μm pore PTFE membrane insert, ADHLSCexerted desirable effects on HSC. This illustrates that at least somesecretion products of ADHLSC, which exert an effect on HSC, do passthrough a filter having pore size of 0.4 μm. Accordingly, in certainembodiments, the conditioned medium may be filtered through a filterhaving pore size not smaller than about 0.4 μm, such as, for example,having pore size between about 0.4 μm and about 1.0 μm or less,preferably between about 0.4 μm and about 0.8 μm or less, morepreferably between about 0.4 μm and about 0.6 μm or less, such as about0.4 μm, e.g., 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, or 0.45 μm.

In certain embodiments, the conditioned medium may be sterilised, suchas conveniently by passing through a microbiological filter having poresize about 0.20 μm, e.g., between 0.20 μm and 0.25 μm, e.g., 0.21 μm,0.22 μm, 0.23 μm, or 0.24 μm.

A further aspect of the invention provides a product derived fromADHLSC-CM, which is a cell-free composition that is obtainable byfractioning ADHLSC-CM (“ADHLSC-CM Fraction”).

Hence, ADHLSC-CM can be used directly for assessing the biologicalactivity or for any appropriate application, but can be used forobtaining a cell-free composition following further processing steps,such as fractionation or any other appropriate processing step that canbe combined to allow obtaining cell-free compositions containing all (orin part) biological molecules secreted by ADHLSC into ADHLSC-CM, inparticular using any appropriate protein separation technique.

The fractionation of ADHLSC-CM can be performed by filtration, fordistinguishing between the components passing through the membrane andthose retained. Depending on the features of the membrane and thedesired composition and/or use, the fraction of interest can be eitherone or the other. For instance, by making use of membranes having a poresize of 0.4 μm, 0.2 μm or less, the fraction passing through themembrane will possibly enriched of soluble proteins and smallermicrovesicles, meanwhile larger microvesicles will be retained by themembrane. Those two fractions can represent distinct cell-freecompositions derived from ADHLSC-CM that can be tested and then used fordifferent applications.

Other means for fractionating ADHLSC-CM involve enzymatically digestingthe ADHLSC-CM (for eliminating specific components of ADHLSC-CM),centrifuging (especially at high speed, for pelleting specificallydesired or undesired components of ADHLSC-CM), adsorbing (by suingheparin or other compound retaining specifically desired or undesiredcomponents of ADHLSC-CM), and/or separating by chromatography (includingimmunoaffinity chromatography, gel chromatography, ion exchange, metalchelate affinity chromatography, HPLC purification and hydrophobicinteraction chromatography), using matrices or beads having specificsize, hydrophobicity, and/or affinity for a ligand.

The ADHLSC-CM and ADHLSC-CM Fractions comprise soluble proteins,together or not with microvesicles. Such soluble proteins form specificcombinations wherein the ratio between proteins and their absolute canvary. However, ADHLSC-CM particularly enriched in a series of growthfactors and cytokines whose alone, or preferably in combinationcomprising part or all of them, contribute defining the basiccomposition and, at least in part, main biological activities ofADHLSC-CM. These soluble proteins are hepatocyte growth factor (HGF),vascular endothelial growth factor (VEGF), eotaxin (CCL11),interleukin-6 (IL-6), and interleukin-8 (IL-8). Each, some or all ofthem are present at a concentration of at least 1 ng/ml. Further solubleproteins that can be present at similar (but or lower concentrations)are selected from matrix metalloproteases (such MMP-2 and MMP-1), growthfactors (such as GM-CSF or PDGF-bb, chemokines (such as RANTES orMIP-1a), and cytokines (such as IFN-gamma, TNF-alpha, IL-10 or otherproteins belonging to the group of interleukins), and other that can beidentified by separating and analysing ADHLSC-CM according to theaffinity, activity, or size (e.g. below 3 kDa, 10 kDa, 20 kDa, or 50kDa) of the proteins. Depending on the actual concentration of suchsoluble proteins within the ADHLSC-CM Fraction, it can be consequentlyused for purifying any of such proteins.

The identification of soluble proteins that are present in ADHLSC-CM andthat characterize one or more of its biological and functional features,can be performed by using the technologies that are commonly applied fordetermining the secretome of a cell type or a cell population, inparallel to the general transcriptome and proteome, as described in theliterature (Eichelbaum K et al. 2012; Mukherjee P and Mani S, 2013). Inparticular, major findings on the composition and activities of ADHLSCsecretome as presented by ADHLSC-CM can be determined in connection toliver activities, as shown in the literature for other types of cellsecretomes for treatments in liver-directed regenerative medicine (Wu Xand Tao R, 2012; Khuu D et al., 2012; Puglisi M et al. 2011). Some ofthe liver-relevant activities of such secretomes that have been shown(at least in vitro) include the decrease of invasiveness of liver tumourcells (Li G et al., 2010; Cavallari C et al., 2013; Qiao L et al., 2008;WO 2011070001), responsiveness to inflammatory serum stimulation (Yagi Het al, 2009), or blocking epithelial-to-mesenchymal transition relevantfor fibrotic activities (Ueno T et al., 2013).

When the ADHLSC-CM, as well as the cell-free compositions that areobtained by fractioning ADHLSC-CM, contains as well (or only)microvesicles, they can be characterized and, when appropriate, selectedaccording their size (below 1, 0.8, 0.4 or 0.2 μm), molecular weight(above 100 kDa, 300 kDa, 500 kDa, or 1000 kDa), and/or composition (interms of proteins, lipids, or nucleic acids) using technologies such asfiltration, (ultra)centrifugation (e.g. at a g-force comprised betweenabout 20,000 and 300,000 g, preferably between about 80,000 and 200,000g) or chromatography.

The term “fraction” as used herein aims to broadly denote the result ofa separation process, in which a mixture (e.g., a solid, liquid, soluteor suspension) is divided up in, i.e., separated into, two or moresmaller quantities (“fractions”) in which the composition changes.Hence, the composition of a fraction is altered compared to, i.e., isdistinct from, the composition of the mixture subjected to thefractionation.

Particularly intended herein are “active” fractions of the conditionedmedium of ADHLSC (“ADHLSC-CM Fractions”), i.e., fractions which at leastpartly retain the desired activity or activities of the conditionedmedium of ADHLSC. For example, an active fraction of ADHLSC-CM mayretain some, e.g., one or more, but not all activities of ADHLSC-CM. Inanother example, an active fraction of ADHLSC-CM may retain allactivities of ADHLSC-CM, but to a lesser degree than ADHLSC-CM. In yetanother example, an active fraction of ADHLSC-CM may retain allactivities of ADHLSC-CM, to the same degree as ADHLSC-CM. Retention of agiven activity or activities in an ADHLSC-CM Fraction may be readilyevaluated by suitable in vitro or in vivo assays.

As intended herein, a “fraction” of ADHLSC-CM may preferably stillcontain a plurality of components. For example, a “fraction” ofADHLSC-CM may preferably contain a plurality of, by means of example andwithout limitation, at least about 5, or at least about 10, or at leastabout 20, or at least about 30, or at least about 40, or at least about50, or at least about 100 or more distinct soluble proteins.

By means of further explanation, any known fractionation methods may beused to fractionate ADHLSC-CM. For example, protein constituents may beseparated from ADHLSC-CM as a protein fraction using standard methods,such as centrifugation and ultracentrifugation to remove membraneousstructures and large nucleic acids, and nuclease (DNAse, RNAse)treatment to remove nucleic acids. A fraction enriched for or comprisingmainly (e.g., consisting essentially of or even consisting of) theprotein constituents of the ADHLSC-CM can thereby be obtained. Suchprotein fraction may be further fractionated into two or more proteinsub-fractions, for example based on any one or more of (1) charge (e.g.,fractionation based on isoelectric point using pH graded gels or ionexchange columns), (2) size or molecular weight (e.g., fractionationusing size exclusion chromatography or gel electrophoresis), (3)polarity/hydrophobicity (e.g., fractionation using high performanceliquid chromatography or reversed-phase chromatography), (4) affinity toa substance (e.g., fractionation using immunoaffinity chromatography) or(5) heparin sulphate binding (e.g., fractionation using a heparincolumn). As intended herein, a fraction of ADHLSC secretion proteins maypreferably still contain a plurality of components, e.g., a plurality ofsecretion proteins.

Such fractioning may thus comprise applying one or more technologiesknown in the art, such as for example filtering, enzymaticallydigesting, centrifuging, adsorbing, and/or separating by chromatography,to ADHLSC-CM.

Hence, also provided is an isolated fraction of the conditioned mediumof ADHLSC, said fraction having composition distinct from thecomposition of the conditioned medium. A related aspect provides amethod for producing the fraction of the conditioned medium of ADHLSCcomprising isolating the fraction from the conditioned medium.

The term “isolated” with reference to a particular component generallydenotes that such component exists in separation from—for example, hasbeen separated from or prepared and/or maintained in separation from—oneor more other components of its natural environment. The term “isolated”as used herein may preferably also encompass the qualifier “purified”.By means of example, the term “purified” with reference to a substancedoes not require absolute purity. Instead, it denotes that suchsubstance is in a discrete environment in which its abundance(conveniently expressed in terms of mass or weight or concentration)relative to other relevant substances is greater than in the materialwhich was subjected to the purification. A discrete environment denotesa single medium, such as for example a single solution, gel,precipitate, lyophilisate, etc.

For example, microvesicles may be separated from ADHLSC-CM as amicrovesicle fraction using standard methods. Cell-derived microvesicles(MV) are small vesicles released by cells that express thecharacteristic antigens of the cell from which they originate and carrymembrane and cytoplasmic constituents and have been described as a newmechanism of cell communication. MV may contribute to tissueregeneration and repair. MV may be typically spheroid in shape withdiameters within the range of 50 nm to 5 μm, more typically of between0.2 and 1 μm. If the particle is not spheroid in shape, the abovementioned values are referred to the largest dimension of the particle.Microvesicles are shed from almost all cell types that originatedirectly from the plasma membrane of the cell and reflect the antigeniccontent of the cells from which they originate. Microvesicles can play arole in intercellular communication between cells, even at distantsites, and can transport mRNA, miRNA, and proteins that may have variouseffects on cells interacting with them, such as on immunomodulation orproliferation. Methods to isolate MV fraction may includeultracentrifugation, e.g., (repeated) ultracentrifugation of ADHLSC-CMat 100,000 g for 1 h at 4° C., and re-suspending the pellet in asuitable medium or buffer. An ADHLSC-CM fraction enriched for orcomprising mainly (e.g., consisting essentially of or even consistingof) MV of the CM is thereby obtained.

Also provided herein is an isolated ADHLSC-CM Fraction of said mixedsecretion proteins, said fraction having composition distinct from thecomposition of the mixed secretion proteins. The term “mixed secretionproteins” is used herein to conveniently refer to the collection orplurality of protein products of ADHLSC found in ADHLSC-CM.

The terms “peptide” and “polypeptide” refer to compounds made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” as used herein may be synonymous with the terms “peptide” and“polypeptide” (i.e., the latter two terms are within the ambit of theterm “protein”) or may refer, in addition, to a polypeptide (a compoundmade up of a single chain of amino acid residues linked by peptidebonds) complexed with one or more same or other polypeptides. Thecomplex may be held by non-covalent interactions and/or covalentbond(s), e.g., disulfide bonds, between the constituent polypeptides. Inaddition, the term “protein” may refer to peptides, polypeptides orproteins in which one or more amino acid residues are modified bypost-translational modification, including, but not limited toglycosylation, phosphorylation, formation of intra- or inter-moleculardisulphide bonds, and the like.

The phrase “mixed secretion proteins” is not to be deemed as anindication that the collection of proteins would be limited to thosewhich have been secreted by the cells' secretion machinery. Instead, thephrase is intended to cover any cellular proteins that have entered themedium.

However, proteins that have been secreted by the cells' secretionmachinery may constitute prominent and preferred members of thecollection.

A preparation of isolated mixed microvesicles produced by ADHLSCrepresents an ADHLSC-CM Fraction, said fraction having a compositiondistinct from the composition of the initial ADHLSC-CM. The term “mixedmicrovesicles” is used herein to conveniently refer to the collection orplurality of microvesicles produced by ADHLSC, found in ADHLSC-CM, andpreferably isolated as an ADHLSC-CM Fraction.

In certain embodiments, ADHLSC-CM or the ADHLSC-CM Fraction may beconcentrated compared to the medium as directly separated from the cellsused to condition it, or compared to the cell-free compositions that areobtained by fractioning ADHLSC-CM. For example, the products may be soconcentrated at least about 5-fold, or at least about 10-fold, or atleast about 15-fold, or at least about 20-fold, or at least about25-fold, or at least about 50-fold, or at least about 100-fold. Anysuitable technique may be employed to eliminate the excess of liquidcomponents and concentrate the ADHLSC-CM or an ADHLSC-CM Fraction, suchas without limitation ultrafiltration, evaporation, dialysis,lyophilisation, and the like. For example, the liquid products may beconcentrated using ultrafiltration units for centrifugation withmembranes having very small pore size and/or weight cut-off. Protein andother soluble components of the liquid products may be concentrated byusing ultrafiltration units with a 3 kD molecular weight cut-off (AmiconUltra-PL 3, Millipore, Bedford, Mass., USA). The liquid product that isconcentrated by ultrafiltration retains most of its proteins and generalbiological activities due to its components not passing through thefilter. Otherwise, by using larger cut-off and/or preliminarydifferential centrifugation steps, the concentration may be associatedto a further fractionation of ADHLSC-CM or an ADHLSC-CM Fraction (e.g.by eliminating components of lower molecular size and weight).

In certain embodiments, ADHLSC-CM or an ADHLSC-CM Fraction, may bediluted compared to the medium as directly separated from the cells usedto condition it, or compared to the cell-free compositions that areobtained by fractioning ADHLSC-CM. For example, the products may be sodiluted at least about 5-fold, or at least about 10-fold, or at leastabout 15-fold, or at least about 20-fold, or at least about 25-fold. Bymeans of example, the products may be so diluted not more than about50-fold, e.g., not more than about 40-fold or not more than about30-fold. Hence, by means of example the products may be so dilutedbetween about 5-fold and about 50-fold, or between about 10-fold andabout 40-fold, or between about 20-fold and about 30-fold, e.g., about25-fold. The liquid products may be suitably diluted by admixing it withan aqueous solvent, e.g., distilled water, physiological solution (0.90%w/v of NaC), medium, buffer, and the like. The dilution, or thereconstitution of a previously lyophylized or otherwise (cryo-)preservedpreparation of liquid products, can be performed by adding the requiredamount of non-conditioned media or any appropriate cell-free solution,such as a buffer (e.g. PBS) or other physiological solutions that arecompatible with further uses (e.g. administration in a subject).

Hence, at any moment before, during, or after fractionation, the desiredconcentration of the soluble proteins and/or microvesicles withinADHLSC-CM or an ADHLSC-CM Fraction can be obtained by appropriatelyconcentrating (or diluting) said preparation at least about 2-fold, atleast about 5-fold, at least about 10-fold, at least about 20-fold, atleast about 50-fold, at least about 100-fold, or in any appropriateintermediate manner (e.g. at least about 3-fold, 7-fold, 15-fold,25-fold, 30-fold, or 75-fold). If needed, concentration and dilutionsteps may be alternated in order to obtain a product from ADHLSC-CM thatpresent the appropriate activity, volume, and/or composition for furtheruse.

Provided are also any of the aforementioned products, in particularADHLSC-CM or an ADHLSC-CM Fraction, or their combinations with otherproduct, which are suitable for therapeutic uses. Hence, an aspectprovides the ADHLSC-CM or ADHLSC-CM Fractions, or any other suitable(combination of) product(s) disclosed herein, including combinations ofsuch product(s) with ADHLSC or any other cell preparation that can beadministered for obtaining a therapeutic activity, for use as amedicament. Given the diversity of useful actions of the products thatare derived from ADHLSC-CM, such as their trophic, immuno-modulatory,and/or anti-fibrotic actions, they can provide benefits in a variety ofmedical indications, examples of which are set forth elsewhere in thisspecification.

If such ADHLSC-CM or ADHLSC-CM Fractions are tested initially withoutincluding other exogenous compounds, the addition of such compounds maybe appropriate to have improved (if not synergistic) therapeutic effectat specific concentrations. A list of exogenous compounds being activeingredients that can be combined with ADHLSC-CM or an ADHLSC-CM Fractioninclude cells (in particular having liver origin and/or mesenchymalfeature, such as ADHLSC or other cells suitable for ex vivo or in vivoapplications), proteins (e.g., matrix metalloproteases, growth factors,chemokines, cytokines, hormones, antigens, or antibodies), nutrients(e.g., sugars or vitamins) and/or chemicals (e. g., drugs withimmunomodulating, anti-fibrotic, antiviral or other therapeuticproperties) that were not initially present in ADHLSC-CM or in anADHLSC-CM Fraction and that are known to be effective as medicament fora given indication. The use of specific animal models and clinical data,as well as the understanding of the underlying biological mechanism, maycontribute in defining which additional compounds can be added, at whichconcentration and, eventually, using which method or schedule ofadministration.

Accordingly, such medical, e.g., prophylactic or therapeutic, uses mayinvolve using ADHLSC-CM, an ADHLSC-CM Fraction, or other suitable(combination of) product(s) as disclosed herein alone or in combinationwith one or more exogenous active ingredients, which may be suitablyadded.

The ADHLSC-CM or the ADHLSC-CM Fraction may be suitably formulated aspharmaceutical formulations comprising a pharmaceutically effectiveamount of any ADHLSC-CM or an ADHLSC-CM Fraction.

Accordingly, a further aspect provides pharmaceutical formulationscomprising a pharmaceutically effective amount of ADHLSC-CM or anADHLSC-CM Fraction or other suitable (combination of) product(s) asdisclosed herein. The pharmaceutical formulations may optionally alsofurther comprise a pharmaceutically effective amount of one or moreexogenous active ingredients, which may be of the type discussed above,e.g., the cells, proteins, nutrients, and/or chemicals.

The pharmaceutical formulations may comprise one or morepharmaceutically acceptable excipients. The pharmaceutical formulationsmay be conveniently formulated into compositions or kits of parts.

The pharmaceutical formulations comprising a pharmaceutically effectiveamount of ADHLSC-CM or an ADHLSC-CM Fraction, may contain excipients andother compounds that allow the correct preservation, stability, oradministration of the pharmaceutical composition. Moreover, a furtherpharmaceutically effective amount of one or more exogenous activeingredients (cells, proteins, nutrients and/or chemicals) may beprovided in the pharmaceutical formulation according to the preliminaryanalysis defined as briefly described above.

The term “pharmaceutically acceptable” as used herein is consistent withthe art and means compatible with the other ingredients of apharmaceutical composition and not deleterious to the recipient thereof.

As used herein, “carrier” or “excipient” includes any and all solvents,diluents, buffers (such as, e.g., neutral buffered saline or phosphatebuffered saline), solubilisers, colloids, dispersion media, vehicles,fillers, chelating agents (such as, e.g., EDTA or glutathione), aminoacids (such as, e.g., glycine), proteins, disintegrants, binders,lubricants, wetting agents, emulsifiers, sweeteners, colorants,flavourings, aromatisers, thickeners, agents for achieving a depoteffect, coatings, antifungal agents, preservatives, antioxidants,tonicity controlling agents, absorption delaying agents, and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active substance, its use in the therapeuticcompositions may be contemplated.

Illustrative, non-limiting carriers for use in formulating thepharmaceutical compositions include, for example, oil-in-water orwater-in-oil emulsions, aqueous compositions with or without inclusionof organic co-solvents suitable for intravenous (IV) use or forinjections in other locations, liposomes or surfactant-containingvesicles, microspheres, microbeads and microsomes, powders, tablets,capsules, suppositories, aqueous suspensions, aerosols, and othercarriers apparent to one of ordinary skill in the art.

Pharmaceutical compositions as intended herein, such as in particularthe ADHLSC-CM or an ADHLSC-CM Fraction, can be formulated foressentially any route of administration, such as without limitation,oral administration (such as, e.g., oral ingestion or inhalation),intranasal administration (such as, e.g., intranasal inhalation orintranasal mucosal application), parenteral administration (such as,e.g., subcutaneous, intravenous, intramuscular, intrahepatic,intrasplenic, intraperitoneal or intrasternal injection or infusion),transdermal or transmucosal (such as, e.g., oral, sublingual,intranasal) administration, topical administration, rectal, vaginal orintra-tracheal instillation, and the like. In this way, the therapeuticeffects attainable by the methods and compositions of the invention canbe, for example, systemic, local, tissue-specific, etc., depending ofthe specific needs of a given application of the invention.

For example, for oral administration, pharmaceutical compositions may beformulated in the form of pills, tablets, lacquered tablets, coated(e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules,aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.In an example, without limitation, preparation of oral dosage forms maybe is suitably accomplished by uniformly and intimately blendingtogether a suitable amount of the active compound in the form of apowder, optionally also including finely divided one or more solidcarrier, and formulating the blend in a pill, tablet or a capsule.Exemplary but non-limiting solid carriers include calcium phosphate,magnesium stearate, talc, sugars (such as, e.g., glucose, mannose,lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin,starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes andion exchange resins. Compressed tablets containing the pharmaceuticalcomposition can be prepared by uniformly and intimately mixing theactive ingredient with a solid carrier such as described above toprovide a mixture having the necessary compression properties, and thencompacting the mixture in a suitable machine to the shape and sizedesired. Molded tablets maybe made by molding in a suitable machine, amixture of powdered compound moistened with an inert liquid diluent.Suitable carriers for soft gelatin capsules and suppositories are, forexample, fats, waxes, semisolid and liquid polyols, natural or hardenedoils, etc.

For example, for oral or nasal aerosol or inhalation administration,pharmaceutical compositions may be formulated with illustrativecarriers, such as, e.g., as in solution with saline, polyethylene glycolor glycols, DPPC, methylcellulose, or in mixture with powdereddispersing agents, further employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilising or dispersing agents known inthe art. Suitable pharmaceutical formulations for administration in theform of aerosols or sprays are, for example, solutions, suspensions oremulsions of the compounds of the invention or their physiologicallytolerable salts in a pharmaceutically acceptable solvent, such asethanol or water, or a mixture of such solvents. If required, theformulation can also additionally contain other pharmaceuticalauxiliaries such as surfactants, emulsifiers and stabilizers as well asa propellant.

Illustratively, delivery may be by use of a single-use delivery device,a mist nebuliser, a breath-activated powder inhaler, an aerosolmetered-dose inhaler (MDI) or any other of the numerous nebuliserdelivery devices available in the art. Additionally, mist tents ordirect administration through endotracheal tubes may also be used.

Examples of carriers for administration via mucosal surfaces depend uponthe particular route, e.g., oral, sublingual, intranasal, etc. Whenadministered orally, illustrative examples include pharmaceutical gradesof mannitol, starch, lactose, magnesium stearate, sodium saccharide,cellulose, magnesium carbonate and the like, with mannitol beingpreferred. When administered intranasally, illustrative examples includepolyethylene glycol, phospholipids, glycols and glycolipids, sucrose,and/or methylcellulose, powder suspensions with or without bulkingagents such as lactose and preservatives such as benzalkonium chloride,EDTA. In a particularly illustrative embodiment, the phospholipid 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is used as an isotonicaqueous carrier at about 0.01-0.2% for intranasal administration of thecompound of the subject invention at a concentration of about 0.1 to 3.0mg/ml.

For example, for parenteral administration, pharmaceutical compositionsmay be advantageously formulated as solutions, suspensions or emulsionswith suitable solvents, diluents, solubilisers or emulsifiers, etc.Suitable solvents are, without limitation, water, physiological salinesolution or alcohols, e.g. ethanol, propanol, glycerol, in addition alsosugar solutions such as glucose, invert sugar, sucrose or mannitolsolutions, or alternatively mixtures of the various solvents mentioned.The injectable solutions or suspensions may be formulated according toknown art, using suitable non-toxic, parenterally-acceptable diluents orsolvents, such as mannitol, 1,3-butanediol, water, Ringer's solution orisotonic sodium chloride solution, or suitable dispersing or wetting andsuspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid.The agents and pharmaceutically acceptable salts thereof of theinvention can also be lyophilised and the lyophilisates obtained used,for example, for the production of injection or infusion preparations.For example, one illustrative example of a carrier for intravenous useincludes a mixture of 10% USP ethanol, 40% USP propylene glycol orpolyethylene glycol 600 and the balance USP Water for Injection (WFI).Other illustrative carriers for intravenous use include 10% USP ethanoland USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2%dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene orparenteral vegetable oil-in-water emulsion. Illustrative examples ofcarriers for subcutaneous or intramuscular use include phosphatebuffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1%triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol andthe balance an acceptable isotonic solution such as 5% dextrose or 0.9%sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USPWFI and 1 to 10% squalene or parenteral vegetable oil-in-wateremulsions.

Where aqueous formulations are preferred, such may comprise one or moresurfactants. For example, the composition can be in the form of amicellar dispersion comprising at least one suitable surfactant, e.g., aphospholipid surfactant. Illustrative examples of phospholipids includediacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol(DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoylphosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such asdimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine(DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidicacids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoylphosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); anddiacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidylethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) anddistearoyl phosphatidyl ethanolamine (DSPE). Typically, asurfactant:active substance molar ratio in an aqueous formulation willbe from about 10:1 to about 1:10, more typically from about 5:1 to about1:5, however any effective amount of surfactant may be used in anaqueous formulation to best suit the specific objectives of interest.

When rectally administered in the form of suppositories, theseformulations may be prepared by mixing the compounds according to theinvention with a suitable non-irritating excipient, such as cocoabutter, synthetic glyceride esters or polyethylene glycols, which aresolid at ordinary temperatures, but liquidify and/or dissolve in therectal cavity to release the drug.

Suitable carriers for microcapsules, implants or rods are, for example,copolymers of glycolic acid and lactic acid.

One skilled in this art will recognize that the above description isillustrative rather than exhaustive. Indeed, many additionalformulations techniques and pharmaceutically-acceptable excipients andcarrier solutions are well-known to those skilled in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

The dosage or amount of the present active substances used, optionallyin combination with one or more other active compound to beadministered, depends on the individual case and is, as is customary, tobe adapted to the individual circumstances to achieve an optimum effect.Thus, it depends on the nature and the severity of the disorder to betreated, and also on the sex, age, body weight, general health, diet,mode and time of administration, and individual responsiveness of thehuman or animal to be treated, on the route of administration, efficacy,metabolic stability and duration of action of the compounds used, onwhether the therapy is acute or chronic or prophylactic, or on whetherother active compounds are administered in addition to the agent(s) ofthe invention.

Without limitation, depending on the type and severity of the disease, atypical daily dosage of a product as disclosed herein might range fromabout 1 μg/kg to 1 g/kg of body weight or more, depending on the factorsmentioned above. For instance, a daily dosage of a product as disclosedherein may range from about 1 mg/kg to 1 g/kg of body weight. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. Thus, one or more doses of about 10.0 mg/kg,20.0 mg/kg, 50.0 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, or500 mg/kg of body weight (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g., every day, every other day, every week or everytwo or three weeks.

In certain embodiments, where the active ingredient comprises orconsists of microvesicles, the pharmaceutical formulation may be in adosage form suitable for administration of the microvesicles in anamount from about 1 to about 500 μg/kg, from about 10 to about 250μg/kg, or from about 30 to about 120 μg/kg body weight.

The products as disclosed herein may be used alone or in combinationwith any other active ingredient useful in treating the condition beingtargeted (“combination therapy”). Combination therapies as contemplatedherein may comprise the administration of at least one active substanceof the present invention and at least one other pharmaceutically orbiologically active ingredient.

Said present active substance(s) and said pharmaceutically orbiologically active ingredient(s) may be administered in either the sameor different pharmaceutical formulation(s), simultaneously orsequentially in any order.

The present products and pharmaceutical compositions are useful fortreating patients. The terms “subject” or “patient” are usedinterchangeably and refer to animals, preferably warm-blooded animals,more preferably vertebrates, even more preferably mammals, still morepreferably primates, and most preferably human patients. The term“mammal” includes any animal classified as such, including, but notlimited to, humans, domestic and farm animals, zoo animals, sportanimals, pet animals, companion animals and experimental animals, suchas, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs,cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes.Particularly preferred are human subjects, including both genders andall age categories thereof. Non-human animal subjects may also includeprenatal forms of animals, such as, e.g., embryos or foetuses. Humansubjects may also include foetuses, but by preference not embryos.

As used herein, a phrase such as “a subject in need of treatment”includes subjects that would benefit from treatment of a givencondition. Such subjects may include, without limitation, those thathave been diagnosed with said condition, those prone to develop saidcondition and/or those in whom said condition is to be prevented.

The terms “treat” or “treatment” encompass both the therapeutictreatment of an already developed disease or condition, such as thetherapy of an already developed proliferative disease, as well asprophylactic or preventive measures, wherein the aim is to prevent orlessen the chances of incidence of an undesired affliction, such as toprevent occurrence, development and progression of proliferativediseases. Beneficial or desired clinical results may include, withoutlimitation, alleviation of one or more symptoms or one or morebiological markers, diminishment of extent of disease, stabilised (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, and thelike. “Treatment” can also mean prolonging survival as compared toexpected survival if not receiving treatment.

The term “pharmaceutically effective amount” refers to an amount of anactive compound or pharmaceutical agent that achieves a medicinalbenefit in a subject, e.g., a prophylactic or therapeutic benefit.Hence, “prophylactically effective amount” refers to an amount of anactive compound or pharmaceutical agent that inhibits or delays in asubject the onset of a disorder as being sought by a researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” as used herein, refers to an amountof active compound or pharmaceutical agent that elicits the biologicalor medicinal response in a subject that is being sought by a researcher,veterinarian, medical doctor or other clinician, which may include interalia alleviation of the symptoms of the disease or condition beingtreated. Methods are known in the art for determining therapeuticallyand prophylactically effective doses for the present products.

ADHLSC-CM and ADHLSC-CM Fractions may exert valuable therapeutic effectsin a plurality of pathologies. In particular but without limitation, thetrophic actions (e.g., regulation of cell survival, proliferation,growth and differentiation), the immuno-modulatory actions, and/or theanti-fibrosis actions of the products may play prominent roles.

The ADHLSC-CM or an ADHLSC-CM Fraction can be administeredintravenously, (e.g. intraportally), intrahepatically, intrasplenically,or intraperitoneally, simultaneously or separately from othermedicaments, as well as using a delivery system comprising a source ofthe selected cell-free composition together with a dispenser operable todeliver the conditioned medium to a target tissue or organ. The deliverysystem may involve culturing ADHLSC in an extracorporeal bioreactor (orany other appropriate device) comprising a fluid treatment compartmentand a cell culture compartment, and a selectively permeable barrier(e.g. a membrane, an ultrafiltration cartridge or a bundle of hollowfibers) that separates the fluid treatment compartment and the cellcompartment, wherein the cell culture compartment comprises ADHLSC. Thissystem may further comprise a biological fluid inlet and a biologicalfluid outlet, wherein the biological fluid inlet and outlet permit fluidcommunication between the fluid treatment compartment and a bloodstreamof the subject.

The ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable (combinationof) product(s) as disclosed herein, can be used in the treatment (as aprophylaxis or a therapy) of a series of disorders such as fibroticdisorders, liver disorders, organ injury or failure, or any other or thepathological disruption, inflammation, degeneration, and/orproliferation within an organ or a tissue. The ADHLSC-CM or of thecell-free compositions that are obtained by fractioning ADHLSC-CM canautologous to the subject (if ADHLSC from the same subject were used forproducing ADHLSC-CM) or allogeneic to the subject (if ADHLSC fromanother subject were used for producing ADHLSC-CM).

Moreover, ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable(combination of) product(s) as disclosed herein, can be used assupporting the transplantation of an organ (in particular liver) orcells (in particular, transplantation of ADHLSC, hepatocytes, or organ-and/or differentiation-specific MSC), as a subsidiary treatment that canbe administered before, after, or when performing transplantation.

The ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable (combinationof) product(s) as disclosed herein, can be used in the treatment oforgan injury or organ failure, preferably wherein the organ is liver, orfor use in the treatment of excessive accumulation of fibrous tissue,preferably wherein excessive accumulation of fibrous tissue affects theliver, or for use in the treatment of a liver disease, or for use in thetreatment of a proliferative disease, preferably wherein theproliferative disease affects the liver.

ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable (combination of)product(s) as disclosed herein, may be also used for preventing ortreating liver disorders, in particular those disorders involving thepathological disruption, inflammation, degeneration, and/orproliferation of liver cells, such as liver fibrotic disorders, liverfailure, acute or chronic liver failure, acute liver infections,cholangitis, biliary cirrhosis. The liver fibrotic disorders may beassociated, or caused by, diabetes, metabolic syndromes, viralhepatitis, chronic persistent or active hepatitis, autoimmune hepatitis,alcoholic liver disease, fatty liver disease, nonalcoholicsteatohepatitis (NASH), acute-on-chronic liver failure (ACLF), primarybiliary cirrhosis, primary sclerosing cholangitis, biliary atresia,congenital liver disease, hepatocellular carcinoma, or liver fibrosiscoincident with chronic or repeated alcohol ingestion, with infection,with liver transplant, or with drug induced liver injury.

The ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable (combinationof) product(s) as disclosed herein, may be administered after that aninitial diagnosis, risk assessment, or progression of the specificdisease is established by analysis of biopsies or on the basis of theelevated level in the plasma of some markers of liver injury,dysfunction, fibrosis, alteration, or necrosis. These biochemicalmarkers associated to liver activity and status can be selected amongthose disclosed in the literature and in particular Alanineaminotransferase (ALAT), Aspartate aminotransfersase (ASAT), AlkalinePhosphatase (AP), Gamma Glutamyl transpeptidase (GGT), Cytokeratin-18(CK-18) or Resistin. In a particular embodiment, the liver disorder is afatty liver disease in which the elevation of one or more of thesemarkers is associated to a more or less significant steatosis in theliver, as it can be confirmed by a liver biopsy.

The ADHLSC-CM or an ADHLSC-CM Fraction, or other suitable (combinationof) product(s) as disclosed herein, can be used for preventing ortreating fibrotic disorders, including but not limited to liver fibroticdisorders, pulmonary fibrosis, kidney fibrosis, prostate fibrosis,breast fibrosis, heart muscle fibrosis and other disorders involving theincrease specific markers of fibrosis, that is any biochemical,serological markers or any other clinical or echographiccharacteristics, that can be correlated with the presence of fibroticdisease. Examples of biochemical and serological markers include, yetare not limited to extracellular matrix components (such as laminin,tenascin, fibronectin, specific types of collagens).

Organ failure as intended herein may for example encompass liverfailure, such as chronic liver failure and acute liver failure. By meansof example and not limitation, liver failure occurs when large parts ofthe liver become damaged and the liver is no longer able to perform itsnormal physiological function. In some aspects, liver failure can bediagnosed using any assay of liver function. In some embodiments, liverfailure can be diagnosed (e.g., initially diagnosed) based on asubject's symptoms. Symptoms that are associated with liver failureinclude, for example, one or more of the following, nausea, loss ofappetite, fatigue, diarrhea, jaundice, abnormal/excessive bleeding(e.g., coagulopathy), swollen abdomen, mental disorientation orconfusion (e.g., hepatic encephalopathy), sleepiness, and coma. Chronicliver failure occurs over months to years and is most commonly caused byviruses (e.g., HBV and HCV), long-term/excessive alcohol consumption,cirrhosis, hemochromatosis, and malnutrition.

Acute liver failure is the appearance of severe complications after thefirst signs of liver disease (e.g., jaundice). Acute liver failureincludes a number of conditions, all of which involve severe hepatocyteinjury or necrosis. In most cases of acute liver failure, massivenecrosis of hepatocytes occurs; however, hepatocellular failure withoutnecrosis is characteristic of fatty liver of pregnancy and Reye'ssyndrome. Altered mental status (hepatic encephalopathy) andcoagulopathy in the setting of a hepatic disease generally define acuteliver failure. Consequently, acute liver failure is generally clinicallydefined as the development of coagulopathy, usually an internationalnormalized ratio (a measure of the time it takes blood to clot comparedto an average value—INR) of greater than 1.5, and any degree of mentalalteration (encephalopathy) in a patient without preexisting cirrhosisand with an illness of less than 26 weeks' duration. Acute liver failure(or acute-on-chronic liver failure) indicates that the liver hassustained severe damage resulting in the dysfunction of 80-90% of livercells.

Acute liver failure occurs when the liver fails rapidly. Hyperacuteliver failure is characterized as failure of the liver within one week.Acute liver failure is characterized as the failure of the liver within8-28 days. Subacute liver failure is characterized as the failure of theliver within 4-12 weeks. In some embodiments, the compositions andmethods described herein are particularly suitable for the treatment ofhyperacute, acute, and subacute liver failure, all of which are referredto herein as “acute liver failure.” Common causes for acute liverfailure include, for example, viral hepatitis, exposure to certain drugsand toxins (e.g., fluorinated hydrocarbons (e.g., trichloroethylene andtetrachloroethane), Amanita phalloides (e.g., commonly found in the“death-cap mushroom”), acetaminophen (paracetamol), halothanes,sulfonamides, henytoins), cardiac-related hepatic ischemia (e.g.,myocardial infarction, cardiac arrest, cardiomyopathy, and pulmonaryembolism), renal failure, occlusion of hepatic venous outflow (e.g.,Budd-Chiari syndrome), Wilson's disease, acute fatty liver of pregnancy,amebic abscesses, and disseminated tuberculosis.

Acute liver failure encompasses both fulminant hepatic failure (FHF) andsub-fulminant hepatic failure (or late-onset hepatic failure). FHF isgenerally used to describe the development of encephalopathy within 8weeks of the onset of symptoms in a patient with a previously healthyliver. Sub-fulminant hepatic failure is reserved for patients with liverdisease for up to 26 weeks prior to the development of hepaticencephalopathy.

FHF is usually defined as the severe impairment of hepatic functions inthe absence of pre-existing liver disease. FHF may result from exposureof a susceptible individual to an agent capable of producing serioushepatic injury. Examples of such agents include infectious agents,excessive alcohol, hepatotoxic metabolites, and hepatotoxic compounds(e.g., drugs). Other causes include congenital abnormalities, autoimmunedisease, and metabolic disease. In many cases the precise etiology ofthe condition is unknown (e.g., idiopathic). FHF may be diagnosed, forexample, using the liver function assays described above.

By means of another example, Multiple Organ Failure is generally definedas parenchymal cell loss associated with a local and systemicinflammatory response. More specifically, organ failure is the failureof an essential system in the body requiring medical intervention.Multiple organ dysfunction syndrome (MODS) is altered organ function inan acutely ill patient requiring medical intervention to performhomeostasis. MODS usually involves two or more organs.

MODS typically results from infection, injury (accident, surgery),hypoperfusion and hypermetabolism. Following an initiating event, anuncontrolled inflammatory response ensues, which causes tissue injuryand triggers local and systemic responses. Respiratory failure is commonin the first 72 hours after the original insult, hepatic failure iscommon in the first 5-7 days, gastrointestinal bleeding may occur at10-15 days, and renal failure is common at 11-17 days. Mortality ratesfor MODS vary from 30% to 100%. There is currently no effectivetherapeutic regimen available to reverse established MODS.

By means of example and not limitation, liver fibrosis is the excessiveaccumulation of extracellular matrix proteins including collagen thatoccurs in most types of chronic liver diseases. Advanced liver fibrosisresults in cirrhosis, liver failure, and portal hypertension, and oftenrequires liver transplantation. A key event in the etiology of liverfibrosis is inappropriate or excessive hepatic stellate cell activation.

The term “liver disease” applies to many diseases and disorders thatcause the liver to function improperly or to cease functioning, and thisloss of liver function is indicative of liver disease.

Thus, assays of liver function are frequently used to diagnose liverdisease. Examples of such assays include, but are not limited to, thefollowing:

(1) Assays to determine the levels of serum enzymes such as lactatedehydrogenase (LDH), alkaline phosphatase (ALP), aspartateaminotransferase (AST), and alanine aminotransferase (ALT), where anincrease in enzyme levels indicates liver disease. One of skill in theart will reasonably understand that these enzyme assays indicate onlythat the liver has been damaged. They do not assess the liver's abilityto function. Other tests can be used to assay a liver's ability tofunction.

(2) Assays to determine serum bilirubin levels. Serum bilirubin levelsare reported as total bilirubin and direct bilirubin. Normal values oftotal serum bilirubin are 0.1-1.0 mg/dL (e.g., about 2-18 mmol/L).Normal values of direct bilirubin are 0.0-0.2 mg/dL (0-4 mmol/L).Increases in serum bilirubin are indicative of liver disease.

(3) Assays to determine serum protein levels, for example, albumin andthe globulins (e.g., alpha, beta, gamma). Normal values for total serumproteins are 6.0-8.0 g/dl (60-80 g/L). A decrease in serum albumin isindicative of liver disease. An increase in globulin is indicative ofliver disease.

Other tests include prothrombin time, international normalized ratio,activated clotting time (ACT), partial thromboplastin time (PTT),prothrombin consumption time (PCT), fibrinogen, coagulation factors;alpha-fetoprotein, and alpha-fetoprotein-L3 (percent).

In another aspect ADHLSC-CM or an ADHLSC-CM Fraction, or thepharmaceutical formulation comprising them, or combinations thereof, maybe for use in the treatment of a proliferative disease.

The term “proliferative disease or disorder” generally refers to anydisease or disorder characterized neoplastic cell growth andproliferation, whether benign, pre-malignant, or malignant The termproliferative disease generally includes all transformed cells andtissues and all cancerous cells and tissues. Proliferative diseases ordisorders include, but are not limited to abnormal cell growth, benigntumours, premalignant or precancerous lesions, malignant tumours, andcancer.

Examples of proliferative diseases and/or disorders are benign,pre-malignant, and malignant neoplasms located in any tissue or organ,such as in the prostate, colon, abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous (central and peripheral), lymphatic system, pelvic, skin, softtissue, spleen, thoracic, or urogenital tract. An organ that may bepreferably affected is the liver.

Further subject matter covered by the aspects of the invention includesany one or a combination of two or more of ADHLSC (or other suitabletype of cell such as primary hepatocytes or a suitable stem orprogenitor cells of liver or other origin), ADHLSC-CM or an ADHLSC-CMFraction, or other suitable (combination of) product(s) as disclosedherein, or the pharmaceutical formulation as disclosed herein, for usein inhibiting fibrogenesis and fibrosis in vivo.

Inhibition of fibrogenesis and fibrosis in vivo may be advantageouslyemployed in the treatment of fibrotic diseases, which are commonlytypified by excessive accumulation of fibrous tissue to the detriment ofparenchymal cells, and therefore by reduced function due to the loss ofparenchymal cells.

Also provided is use of any one or a combination of two or more ofADHLSC, ADHLSC-CM, an ADHLSC-CM Fraction, or other suitable (combinationof) product(s) as disclosed herein, or the pharmaceutical formulation asdisclosed herein, for inhibiting fibrogenesis in vitro. Also providedare corresponding methods of inhibiting fibrogenesis in vitro.

Such combination of biological products can be tested in a culturesystem making use of HSC; using ADHLSC-based co-culture systems like theone described in the Examples as a reference, e.g., the co-culture in aTranswell® system, wherein the cells are separated by a semipermeablemembrane that prevents the exchanges of cells, but allows mostextracellular components to pass.

As set forth in the experimental section, the inventors demonstratedthat in a co-culture system where ADHLSC were separated from HSC by a0.4 μm pore PTFE membrane insert, ADHLSC exerted desirable effects onHSC. This illustrates at least how such combination products can becompared to the secretion products of ADHLSC that exert an effect onHSC, by passing through a filter having pore size of 0.4 μm, inparticular obtaining an ADHLSC-CM fraction containing microvesicles thatare smaller than 0.4 μm.

The terms “hepatic stellate cells” or “HSC” are well-understood in thefield. By means of further guidance, these terms denote non-parenchymalliver cells which reside in the space of Disse within the hepaticmicrocirculatory unit, and can be distinguished using intravitalfluorescent microscopy (IVFM) due to the autofluorescence from theirintracellular vitamin A.

Also provided is a pharmaceutical formulation comprising HSC and any oneor a combination of two or more of ADHLSC, ADHLSC-CM or the cell-freecompositions that are obtained by fractioning ADHLSC-CM, or othersuitable product(s) as disclosed herein. The pharmaceutical formulationmay further comprise hepatocytes, e.g., primary hepatocytes orhepatocytes differentiated in vitro from a suitable progenitor cells,e.g., MSC or ADHLSC.

Further provided is a pharmaceutical formulation comprising hepatocytesand any one or a combination of two or more of ADHLSC, ADHLSC-CM or thecell-free compositions that are obtained by fractioning ADHLSC-CM, orother suitable product(s) as disclosed herein.

These pharmaceutical formulations may be suitably formulated asdescribed in detail above. Composition of pharmaceutical formulationcomprising live cells may impose certain requirements, such as forexample, the compositions may comprise a suitable buffer system (e.g.,phosphate or carbonate buffer system) to achieve desirable pH, moreusually near neutral pH, and may comprise sufficient salt to ensureisoosmotic conditions for the cells to prevent osmotic stress. Forexample, suitable solution for these purposes may be phosphate-bufferedsaline (PBS), sodium chloride solution, Ringer's Injection or LactatedRinger's Injection, as known in the art. Further, the compositions maycomprise a carrier protein, e.g., albumin, which may increase theviability of the cells. Such requirements are well appreciated by theskilled person Where pharmaceutical formulations as formulatedthroughout this specification comprise cells, such as without limitationhepatocytes or ADHLSC, or other stem or progenitor cells of liver (orother) origin, the cells may be administered to human subjects withoutlimitation in doses ranging from about 1×10⁵ to 1×10¹² cells, such aspreferably doses of about 1×10⁶ to 1×10¹⁰ cells, or doses of about 1×10⁷to about 1×10⁹ cells, e.g., about 1×10⁷, about 1×10⁸, or about 1×10⁹cells. However, the precise determination of a therapeutically effectivedose may be based on factors individual to each patient, including theirsize, age, size tissue damage, and amount of time since the damageoccurred, and can be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art.

Such pharmaceutical formulations may be particularly useful for thetreatment of diseases discussed elsewhere in this specification, moreparticularly liver diseases. More particularly, pharmaceuticalformulations comprising cells may be intended for transplantation intothe liver.

Additionally, ADHLSC-CM can be used methods for identifying abiologically active compound for the treatment of a liver disease, themethod comprising;

(a) obtaining one or more fractions of a ADHLSC-CM:

(b) assaying the ability of one or more of the fractions to increase ordecrease one or more biological activities (such as metabolism,proliferation, survival, activation, apoptosis, migration, engraftment,or differentiation) of one or more cells found in liver (such ashepatocytes, hepatic stellate cells, liver myofibroblasts, or hepaticsinusoidal cells) in vivo, in vitro, and/or ex vivo;

(c) selecting a fraction of the ADHLSC-CM that increase or decrease oneor such activities; and

(d) identifying one or more molecules present in the selected fraction.

The ensuing clauses provide additional illustration of certain aspectsand embodiments that have been disclosed in accordance with the presentdisclosure:

1. A cell-free conditioned medium obtainable by culturing adult-derivedhuman liver stem/progenitor cells (ADHLSC) in a cell culture medium andseparating the cell culture medium from the cells.

2. The cell-free conditioned medium according to clause 1, wherein themedium is serum-free.

3. A cell-free composition obtainable by fractioning the cell-freeconditioned medium according to clause 1 or 2.

4. The cell-free composition according to clause 3, wherein saidfractioning comprises filtering, enzymatically digesting, centrifuging,adsorbing, and/or separating by chromatography the cell-free conditionedmedium.

5. The cell-free conditioned medium according to clause 1 or 2, or thecell-free composition according to clause 3 or 4, that contains solubleproteins and/or microvesicles.

6. The cell-free conditioned medium according to any one of clauses 1, 2or 5, or the cell-free composition according to any one of clauses 3 to5, comprising:

-   -   (a) at least one of soluble proteins selected from the group        consisting of: hepatocyte growth factor (HGF), Vascular        endothelial growth factor (VEGF), eotaxin (CCL11), interleukin-6        (IL-6), and interleukin-8 (IL-8); and, optionally    -   (b) at least one of soluble proteins selected from the group        consisting of matrix metalloproteases, growth factors,        chemokines, and cytokines.

7. The cell-free conditioned medium according to clauses 5 or 6, or thecell-free composition according to clauses 3 to 6, wherein the solubleproteins are present at a concentration of at least 1 ng/ml.

8. The cell-free conditioned medium according to any one of clauses 1, 2or 5 to 7, or the cell-free composition according to any one of clauses3 to 7, wherein said microvesicles are selected according their size,molecular weight, and/or composition.

9. The cell-free conditioned medium according to any one of clauses 1,2, or 5 to 8, or the cell-free composition according to any one ofclauses 3 to 8 that is concentrated at least about 5-fold, at leastabout 10-fold, at least about 20-fold, at least about 50-fold, or atleast about 100-fold.

10. The cell-free conditioned medium according to any one of clauses 1,2, or 5 to 8, or the cell-free composition according to any one ofclauses 3 to 8 that is diluted at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, or at leastabout 100-fold.

11. A method for producing a cell-free conditioned medium comprising thesteps of culturing ADHLSC in a cell culture medium and separating thecell culture medium from ADHLSC.

12. The method for producing a cell-free conditioned medium according toclause 11, wherein:

-   -   (a) the cell culture medium is a serum-free medium; and/or    -   (b) the cell culture medium is separated from ADHLSC after        culturing ADHLSC in the cell culture medium for at least 2        hours, at least 4 hours, at least 6 hours, at least 8 hours, at        least 12 hours, or at least 24 hours.

13. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10 or the cell-free composition of any of clauses 3 to 10, for use as amedicament.

14. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10 or the cell-free composition of any of clauses 3 to 10, for use as amedicament in combination with one or more exogenous active ingredients.

15. A pharmaceutical formulation comprising a pharmaceutically effectiveamount of any one of the cell-free conditioned medium of any one ofclauses 1, 2, or 5 to 10 or the cell-free composition of any of clauses3 to 10.

16. A pharmaceutical formulation comprising a pharmaceutically effectiveamount of a combination of any one of the cell-free conditioned mediumof any one of clauses 1, 2, or 5 to 10 and/or the cell-free compositionof any of clauses 3 to 10, and one or more exogenous active ingredients.

17. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10, the cell-free composition of any one of clauses 3 to 10, or thepharmaceutical formulation according to any one of clauses 15 or 16, ora combination of two or more thereof, for use in the treatment of afibrotic disorder.

18. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10, the cell-free composition of any one of clauses 3 to 10, or thepharmaceutical formulation according to any one of clauses 15 or 16, ora combination of two or more thereof, for use in the treatment of aliver disorder.

19. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10, the cell-free composition of any one of clauses 3 to 10, or thepharmaceutical formulation according to any one of clauses 15 or 16, ora combination of two or more thereof, for use in the treatment of organinjury or failure.

20. The cell-free conditioned medium of any one of clauses 1, 2, or 5 to10, the cell-free composition of any one of clauses 3 to 10, or thepharmaceutical formulation according to any one of clauses 15 or 16, ora combination of two or more thereof, for use in organ or celltransplantation.

21. A method of treating a disorder in a subject in need of saidtreatment comprising the administration of a therapeutically orprophylactically effective amount of the cell-free conditioned medium ofany one of clauses 1, 2, 5 to 10, the cell-free composition of any oneof clauses 3 to 10, or the pharmaceutical formulation of clause 15 or16, or of a combination of two or more thereof, to the subject.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing description. Accordingly, it is intendedto embrace all such alternatives, modifications, and variations asfollows in the spirit and broad scope of the appended claims.

The herein disclosed aspects and embodiments of the invention arefurther supported by the following non-limiting examples.

EXAMPLES Example 1—Profile of ADHLSC and HSC Secretome as Established inConditioned Media

Materials & Methods

ADHLSC and HSC Isolation and Culture

The protocol and experiments were approved by the ethical committees ofthe St-Luc Hospital and faculty of Medicine of Université catholique deLouvain. An agreement from the Belgian Ministry of Health was obtainedfor the Hepatocytes and Hepatic Stem Cells Bank. A written and signedinformed consent has been obtained for each human liver used in thecurrent study. Four donors were used in the current study, as shown inTable 1.

TABLE 1 Donor Reason Blood Ischemia number (Age) Gender of death grouptime 89 (3 days) M Respiratory A+ 4 hours 93 (2 years) F Metabolic O+ 1hour 43 min disease (liver transplanted) 97 (7 months) F Meningitis A+ 5hours 30 min 98 (7 days) M Cardio- O− 4 hours 20 min respiratory arrest

ADHLSC were obtained subsequently to primary culture of the liverparenchymal fraction previously obtained after a two-step collagenaseperfusion, filtration and low speed centrifugation, as previouslydescribed (Najimi M et al., 2007; WO2007/071339). HSC were isolated fromthe corresponding non-parenchymal fraction using a Nycodenz® gradientcentrifugation step (Myegaard, Oslo, Norway) as previously described(Guimaraes E et al. 2010).

Both cell types were cultured using DMEM containing 4.5 g/L glucose(Invitrogen) supplemented with 10% v/v Fetal Calf Serum (PAA) and 1%Penicillin/Streptomycin (Invitrogen), at 37° C. in a fully humidifiedatmosphere (5% CO2). When reaching 80% confluence, cells were liftedwith 0.05% trypsin-EDTA (Invitrogen) and re-plated at a density of 5000cells/cm². The viability of recovered cells was evaluated using trypanblue exclusion assay.

Flow cytometry Cells were re-suspended in commercially availableDulbecco's Phosphate-Buffered Saline (D-PBS) at a concentration of 2×10⁵cells/ml. For intracellular immunostaining, cell permeabilization wasperformed with cytofix/cytoperm for 20 minutes at 4° C. (BD Pharmingen).Cells were then washed and incubated for 30 minutes at 4° C. with thefluorescently labelled antibodies (see Table 2). The correspondingcontrol isotypes were used in parallel to evaluate the non-specificbinding. After washing, cells were suspended in Stabilizing Fixative (BDPharmingen) before reading with a CANTO II flow cytometer. The analyseswere performed using the BD FACSDiva Software.

TABLE 2 Ab F CI Suppl. Ref. Conc. Anti-CD29 APC MsIgG1, k BD 559883 1/10Anti-CD44 FITC MsIgG2b, k BD 555478 1/10 Anti-CD45 PE-Cy7 MsIgG1, k BD557748 1/10 Anti-CD73 PE MsIgG1, k BD 550257 1/10 Anti-CD90 APC MsIgG1,k BD 559869 1/10 Anti-CD117 APC MsIgG1, k BD 550412 1/10 Anti-CD133/2 PEMsIgG1 Miltenyi 130-080-901 1/5  (Ab: antibody specificity, F:fluorochrome, CI: corresponding isotype, Suppl.: commercial supplier,Ref.: supplier's reference, Conc.: concentration for flow cytometryanalysis)

Production of Conditioned Media for ADHLSC and HSC (ADHLSC-CM andHSC-CM)

When 60-70% of confluence was reached, the cells were washed and theconditioned medium was replaced by fresh medium without 10% v/v FetalCalf Serum. After 24 hours incubation, supernatants were collected andstored for further assays. Corresponding cells were lifted for countingand viability evaluation. The concentration of secreted protein isexpressed as nanograms (ng) or picograms (pg) that are secreted in 24hours by 10⁵ cells during 24 hours.

ELISA

The ELISA analyses were performed on culture supernatants collected 24hours after incubation with serum-free medium. The growth factors andcytokines concentrations were calculated for 10⁵ cells. The measurementof the absorbance at 450 nm was done with a Victor X4 plate Reader(PerkinElmer).

Collagen secretion analysis was performed by using an ELISA kit for theprocollagen type I C-Peptide (Takara Bio Inc, Japan). Hepatocyte GrowthFactor (HGF) and Transforming Growth Factor beta 1 (TGFβ1) levels in theculture supernatants were assayed by using Quantikine ELISA Kits fromR&D Systems. For HGF and TGFβ1 kits, a reading at 570 nm was subtractedto the 450 nm reading to correct the optical imperfections of theplates. The experiments were performed according to the manufacturer'sinstructions. For TGFβ1 ELISA, samples were activated by acidificationfollowed by neutralization in order to make latent TGFβ1 detectable byusing the Quantikine TGFβ1 immunoassay.

Luminex Analysis

The Bio-Plex Pro Human Cytokine 27-plex Assay kit (including IL-1b,IP-10, IL-2, IL-4, IL-6, IL-7, IL-188 9, IL-10, IL-13, IL-15, Eotaxin,FGF, GM-CSF, interferon-gamma (IFN-γ), MIP-1a, MIP-1b, RANTES, TNFα,IL-1ra, IL-5, IL-8, IL-12, IL-17, G-CSF, MCP-1, PDGF-bb and VEGF;Bio-Rad) and the Luminex technology (Bio-Plex 200, Biorad) was used toinvestigate the secretome of both liver cell types. The principle of thetechnique is based on color-coded beads and enables to detect up to 100cytokines simultaneously. The primary antibody directed against thetarget protein is conjugated with the dyed beads. After several washesto remove unbound proteins, a secondary biotinylated antibody is addedto the reaction. Streptavidin-phycoerythrin (Streptavidin-PE) is thenadded to bind the biotinylated antibody. By measuring the relativefluorescence intensity, the antigen-antibody reaction can be measured.

The assays were performed following the manufacturer's instructions.Briefly, after the pre-wetting of the plate, 50 μl of the beads wereadded in each well and washed twice. 50 g of the samples (serum freeculture supernatants recovered after 24 hours of culture) were added tothe plate. The plate was shaken during 30 seconds and then incubated for45 minutes on a plate shaker at 120 rpm at room temperature. The platewas washed three times with the Bio-Plex wash buffer and 251 of thedetection antibody was added in each well and incubated for 30 minuteson a plate shaker at 120 rpm. The plate was then washed three times withthe Bio-Plex wash buffer and 50 g of the Streptavidin-PE solution wasadded in each well. The plate was shaken during 30 seconds and incubatedfor 10 minutes on a plate shaker at 120 rpm. Finally, after three washesof the plate with the Bio-Plex wash buffer, the beads were re-suspendedwith 125 μl of Bio-Plex Assay Buffer. The plate was read by the Luminexmachine and the data were analyzed using Bio-Plex Manager 6.0.

Statistics

Results are expressed as mean standard error of the mean (SEM).Statistical differences were determined by Student's t test for twogroups' comparison. The statistical significance of the differencesbetween samples or conditions was established with the p values *p<0.05,**p<0.01, and ***p<0.001.

Results

ADHLSC and HSC were isolated in four independent liver donors. For eachof them, HSC and ADHLSC were obtained in parallel and then cultivatedunder the same culture conditions and concomitantly followed. Thefibroblastic morphology displayed by both cell types remained stableover the different studied passages and the population cumulativedoubling was similar for the two cell types. The mesenchymal phenotypeof both cell types was investigated by exploring the expression ofseveral specific appropriate markers using flow cytometry. Both celltypes were immuno-positive for most of the membrane markers widely usedto characterize mesenchymal stem cells. This was the case formesenchymal stem cells markers (such as CD73 and CD90) and extracellularmatrix markers (such as CD29 and CD44) for which expression levels werenot significantly different between ADHLSC and HSC for the analysedpassages. The mesenchymal phenotype of both cell types was alsosupported by the negative expression of hematopoietic markers like CD45,CD117 and CD133 as demonstrated using flow cytometry. This data thusconfirmed the presence of mesenchymal markers in both ADHLSC and HSC asreported before (Kordes C et al., 2007; Kordes C et al., 2013; Najimi Met al., 2007).

The secretome of ADHLSC and HSC across the four donors was partiallyanalysed by using the conditioned medium of cell cultures inprotein-specific immunoassays. The experiments were performed onsupernatants collected 24 hours after incubation with serum free medium,obtaining a conditioned media that is called ADHLSC-CM and HSC-CM,respectively.

The analysis started by detecting proteins known to be expressed byactivated HSC. No significant difference was observed between HSC-CM andADHLSC-CM in the concentration of secreted procollagen type-I C-Peptide(about 130 ng/ml/10⁵ cells/24 hours) and TGFβ1, one of the most powerfulpro-fibrotic cytokines and involved in inflammatory and immune responses(about 90 ng/ml/10⁵ cells/24 hours). A significant difference betweenHSC-CM and ADHLSC-CM is observed in the secretion of hepatocyte growthfactor (HGF), a hepatocyte mitogen with anti-inflammatory properties andhaving crucial physiological functions including organ protection andregeneration. Following liver injury, HGF is known to be secreted bydistant organs such as spleen, lungs and kidneys as well as bysinusoidal cells such as Kupffer cells and HSC. ADHLSC-CM contains aboutthree times more HGF than HSC (FIG. 1A).

The secretion of a larger panel of major growth factors, chemokines andcytokines was performed using a multiplex technology. ADHLSC clearlyappear secreting statistically significant higher levels of VEGF, IFN-g,Eotaxin (CXCL11), IL-8 and IL-10 when compared to HSC, withconcentration superior to 1 ng/ml/10⁵/24 hours in ADHLSC-CM. At lowerabsolute levels (i.e., equal to or below 1 ng/ml/10⁵/24 hours), PDGF-bb,TNF-α, IP-10, IL-5, IL-7, IL-9, IL-12, and IL-13 are also present athigher concentration in ADHLSC-CM (FIGS. 1B and 1C). The statisticallysignificant increased concentration of specific cytokines and growthfactors in ADHLSC-CM can be vary from about 2 times (as for IL-5, IL10and IL-13) up to 10 or even 30 times (as Eotaxin, VEGF, and IL-8) morethan HSC-CM. Other cytokines were either undetectable in both cell types(such as bFGF) being more variable across donors (data not shown).

If such data are compared with those previously determined for theconditioned media obtained from a different liver pluripotent progenitor(HLSC-CM; as described in WO2009/150199) or bone marrow Mesenchymal Stemcells (MSC-CM; as described in WO2009/150199), it is also evident thatthe composition of ADHLSC-CM is qualitatively different also from theseconditioned media, as shown in Table 3.

TABLE 3 ADHLSC-CM HLSC-CM MSC-CM (selected from FIG. 1; (selected fromTable 1 of WO2009/150199; expressed as pg/ml/ Protein expressed aspg/ml/10⁶ cells/24 hours) 10⁵cells/24 hours) Eotaxin 5.1 3.96 ≥14335.6IFN-gamma 38.46 104.73  990 ± 91.8 VEGF 896.9 4961.43 ≥45306.2 TNF-alpha3.7 13.04 362.1 ± 33.3  IL8 4205.64 51.04 ≥28231.7 IP-10 0.0 0.0 867.2 ±184.2 HGF 5719 2.3 936.6 ± 151.9 IL2 0.0 0.0 160.1 ± 14.7 

Even if the proteins cited in Table 3 represent only some of the mostrepresentative examples, the differences between the published data andthose obtained using ADHLSC-CM are also remarkable taking inconsideration the fact that concentration for HLSC-CM and MSC-CM areindicated in WO2009/150199 for 10 times more cells. It is evident thatADHLSC secretome is particularly enriched with a combination of usefulsecreted proteins, such as growth factors, chemokines, and cytokinesthat are present in different ratio and amount than in other knownconditioned media.

Thus, ADHLSC secretome, in general and in particular when specificallyobtained and characterized as ADHLSC-CM, represents a featuredistinguishing ADHLSC not only from HSC but also from other liver ormesenchymal cells and their secretome. These molecular features may havesubstantially distinctive effect on how not only ADHLSC themselvesproliferate, are biologically active, and interact with other cells invivo and in vitro, but also on how ADHLSC may affect biologicalactivities exerted by other cells types within the organism, and inparticular within liver. Indeed, potential beneficial effects of medicalinterest may be provided by ADHLSC-CM, as such or a given fractionthereof, alone, in combination or not with ADHLSC or any otherappropriate cell-based, protein-based, and/or chemical drug-basedtreatment.

Example 2—Effects of ADHLSC on HSC Proliferation and Secretion Profile

Materials & Methods

ADHLSC/HSC Co-Culture Model

The indirect co-culture system (Transwell® COL Collagen-Coated 0.4 μmPore PTFE Membrane Insert) was used. In brief, HSC were seeded in thelower chamber at a density of 10 000 cells/cm² while ADHLSC were placedon the membrane insert, with ADHLSC/HSC ratios (cell #/cell #) of 0/1(control) and 1/100. HSC were collected and analysed at the indicatedtime points.

Assays for Comparing ADHLSC and ADHLSC-CM Effects on HSC

ADHLSC-CM and HSC-CM was obtained as indicated above, by collectingADHLSC and HSC supernatants, respectively, after 24 hours of culture inabsence of serum. HSC were incubated with ADHLSC (using two co-culturesystem), HSC-CM, or ADHLSC-CM for 24 hours. The experiments wereperformed by using ADHLSC, ADHLSC-CM, HSC, and HSC-CM from the samedonor (autologous conditions) but repeated also by combining cells orconditioned media from different donors, obtaining qualitatively similarresults.

CCK-8 biochemical assay was performed by using the Cell Counting Kit—8BioChemika (Fluka; cat. No. 96992). Propidium iodide assay was performedaccording to general literature.

For cell cycle analysis, HSC were lifted with 0.05% trypsin-EDTA(Invitrogen) After 24 hours of co-culture. After centrifugation, cellswere washed twice with PBS and fixed with 700 μl of cold ethanol andincubated for 30 minutes on ice. The cells were washed again with PBSbefore being incubated with a solution containing 100 μg/ml PropidiumIodide (PI, Invitrogen), 0.1 mg/ml RNase (Sigma) and 0.01% Triton X-100(Sigma) for 30 minutes at 37° C. and then for 15 minutes on ice, beforereading with a CANTO II flow cytometer. The analyses were performedusing the BD FACSDiva Software. The different phases of the cell cyclewere determined by measuring the area under curve with the FlowJoSoftware.

For Ki-67 immunocytochemistry, HSC were incubated 24 hours with theconditioned media and then fixed using paraformaldehyde 3.5%, for 15 minat room temperature. Endogenous peroxidase was eliminated using hydrogenperoxide 3.3% for 3 minutes. All steps were performed at roomtemperature. Cells were permeabilized using PBS containing 1% TritonX-100 (Sigma) for 10 minutes. Non-specific immuno-staining was preventedby 1 h incubation in PBS containing 1% Bovine Serum Albumin (Sigma).Thereafter, cells were incubated with Ki67 antibody (Dako) for 1 h.After washing, cells were incubated with secondary antibody(EnVision-Dako) during 30 minutes. Detection was performed after 5minutes incubation with liquid DAB and substrate chromogen (Dako).Counterstaining was performed using Mayer's hematoxylin for 10 minutes.Preparations were then mounted for microscopic analysis (DMIL, Leica,Belgium). For each condition, four different fields were analyzed and atotal of 2500 stained/unstained nuclei were counted using the ImageJSoftware.

Results

Fibrogenesis by activated HSC largely contributes to processes resultingin fibrosis of the liver tissues. HSC are “activated” in vivo duringliver injury, and evolve to myofibroblast-like cells, with consequentincrease in cell proliferation and extracellular matrix proteindeposition. At the structural level, activated HSC lose their bigvitamin A-containing lipid droplets and up-regulate the expression ofsome cell adhesion molecules, as well as the secretion ofpro-inflammatory cytokines. In vitro, the fibrotic part of thisactivation process is mimicked by culturing HSC on plastic culturedishes. The effect of ADHLSC in general (and of ADHLSC-CM in particular)on HSC activation and fibrogenesis can be studied in vitro by making useof indirect co-culture systems wherein HSC and ADHLSC are cultured intwo cell culture chambers communicating only through a membrane having aspecific composition, surface, and pore size.

Since activated HSC are characterized by an increased proliferationrate, the first analyses concerned the proliferation of HSC afterco-culture with ADHLSC. The cell number analysis was performed usingmanual counting after the HSC were collected 24 hours, 4 days and 7 dayspost-seeding. Using cells from 4 different donors, a significantdecrease in the number of HSC was noted. In this experiment, astatistically relevant decrease of the number of HSC was obtainedalready by using a ratio between ADHLSC and HSC of 1/100 and comparingwith HSC cultured in absence of ADHLSC (FIG. 2A). This inhibitory effectat such a low ratio appears initiated in the first 24 hours, because nochange in the index proliferation rate was noticed between all groups ofcells.

This effect was confirmed using the same ratio between ADHLSC and HSCand a CCK-8 biochemical assay, a sensitive calorimetric assay thatallows the determination of the number of viable cells in cellproliferation assays (FIG. 2B).

When the effect on adherent and floating HSC is evaluated, it evidentthat a low ratio ADHLSC/HSC of 1/100 provides already a statisticalrelevant decrease of HSC plating, since the number of floating HSC issignificantly increased (FIG. 3A).

Such experiment is repeated by using only conditioned culture medium ofADHLSC (ADHLSC-CM) in order to evaluate its effect on HSC proliferationand adherence, using the conditioned culture medium of HSC (HSC-CM) as acontrol. The effect of ADHLSC-CM was qualitatively similar to the effectobserved with ADHLSC in the co-culture system, suggesting theinvolvement of soluble factor(s) that are secreted by ADHLSC and passacross the membrane separating the two chambers (FIG. 3B). Using flowcytometry and Propidium Iodide staining, no significant difference incell death induction between the HSC cultivated with ADHLSC-CM and thosecultivated with HSC-CM (data not shown).

In order to investigate the mechanisms involved in the diminution of HSCnumber due to ADHLSC-CM, the HSC plating kinetic in presence of eitherADHLSC-CM or HSC-CM was also analysed by counting the adherent and thefloating cells after 2, 4, 8 and 24 hours post-seeding demonstrating adelay of the HSC plating when cultivated with the ADHLSC-CM whencompared to HSC-CM that is maintained though all the time points (FIGS.4A and 4B).

The effects of ADHLSC-CM or ADHLSC on HSC cell cycle were tested byusing propidium iodide (PI) staining and flow cytometry. An increase inthe number of HSC that are blocked in G0/G1 phase and a decrease of thenumber of HSC in the G2/M phase is clearly observed when HSC areincubated either with ADHLSC in the co-culture system (FIG. 5A) or withADHLSC-CM (FIG. 5B). Such evidences were confirmed by Ki67 staininganalysis using immunocytochemistry. A significant decrease in the numberof stained HSC nuclei after an incubation of 24 hours with ADHLSC-CM incomparison with the HSC incubated with HSC-CM is observed (FIG. 5C).

Altogether, these data demonstrate a clear decrease in the number of HSCobserved after co-culture with ADHLSC or ADHLSC-CM, which could be dueto an inhibition of both plating efficiency and cell proliferation ofHSC. It is important to point out that this effect is obtained bycombining ADHLSC and HSC from different donors, as well as by containingADHLSC-CM directly with HSC, and not using the co-culture system. Thisobservation suggests that a component of ADHLSC-CM that can pass acrossthe selected membrane is actually providing this effect on HSC.Activated HSC are known to secrete collagen type I, one of the majorcomponents of the extracellular matrix. Therefore, influence of ADHLSCon the collagen secretion capacity of HSC by measuring the pro-collagenI (precursor of the collagen type I) secretion was tested using an ELISAassay. The HSC were incubated during 24 hours with ADHLSC or withADHLSC-CM. The culture medium was changed after 24 hours and replaced bya serum-free medium. The supernatants were collected after 24 hours andthe HSC were lifted for counting and viability evaluation. A significantdecrease in the amount of pro-collagen I secreted by HSC afterco-culture with ADHLSC at an ADHLSC/HSC ratio of 1/100 in comparisonwith the control group was observed (FIG. 6A). Moreover, by using amultiplex Luminex assay, it can be demonstrated that the secretion ofHGF, a growth factor known to have anti-fibrotic properties, and IL-6 isincreased in the culture supernatants of HSC that were previouslyincubated during 24 hours with ADHLSC using the co-culture system (FIG.6B; similar data were obtained using ADHLSC-CM). At the same time, thesecreted levels of metalloproteinases MMP1 and MMP2, enzymes areinvolved in the extracellular matrix degradation are also increased inall tested donor by at least 1.5 fold (as determined by using themultiplex technology described above). The combination of all these up-and down-regulation of proteins involved in fibrogenesis suggests thatADHLSC-CM (and in particular the component that can pass through themembrane used in the co-culture system) has a global anti-fibroticeffect on HSC that can be exploited for therapeutic applications, suchas for treating liver fibrotic disorders.

The data above support that ADHLSC-CM (and specific components definedaccording to the method of preparation, the molecular weight or otherrelevant features) can be used as a component in a pharmaceuticalcomposition, or within a method of treatment, where it can be used forproviding a biologically balanced mixture of useful cell secretionproducts, such as growth factors, cytokines, chemokines, and othermediators of physiological activities. ADHLSC-CM can be obtained byADHLSC applying an approach similar to one described in Example 1 as acell-free composition and then further purified and/or concentrated(e.g. by filtration, fractionation, chromatography, enzymatic digestion,centrifugation, absorption, or any combination of them), at the scope ofobtaining ADHLSC-CM preparations that are enriched in all, most, or someof its specific components, including soluble proteins and/or vesicularstructures (e.g. microvesicles having a diameter of below 1 μm (such asparticularly below 1.0 μm), below 0.4 μm, below 0.1 μm, or comprisedbetween 1 μm (such as particularly 1.0 μm) and 0.1 μm, or between 0.4 μmand 0.1 μm), that are released by ADHLSC.

Example 3—In Vivo Models and Pharmaceutical Uses of ADHLSC-CM andDerived Cell-Free Compositions

ADHLSC-CM, cell-free compositions that are derived from ADHLSC-CM, andany other composition that is obtained by fractionating and, if needed,concentrated (or diluted) such compositions may be used in a series ofmodels that are preferably selected on the basis of the known biologicalactivities of progenitor cells that are isolated from adult liver (i.e.ADHLSC), and thus more preferably related to models of human liverdiseases that may be treated by making use of such preparations.

The animals (e.g. rats, rabbits, mice) are selected and eventuallytreated to establish the desired model and then injectedintraperitoneally, intravenously (jugular, femoral, portal, or tailvein), intra-arterially (into aorta via carotid or femoral artery),intrahepatically, or intrasplenically with a given volume (e.g., 0.01-1ml) of ADHLSC-CM, or an equivalent amount of the protein fraction ofADHLSC-CM, or an equivalent amount of the microvesicles fraction ofADHLSC-CM. Suitable controls may include an identical cell culturemedium not conditioned by any cells, or an identical medium conditionedby culturing Hepatic Stellate Cells, Mesenchymal Stem Cells (such asbone marrow MSC) or any other cell type or population, as such or as acorresponding fraction thereof, and alone or in combination with anyother appropriate drug or treatment that is applied simultaneously,previously, or subsequently to the administration of ADHLSC-CM, or anequivalent amount of the protein fraction of ADHLSC-CM, or an equivalentamount of the microvesicles fraction of ADHLSC-CM (including the use ofa cell-based therapy based on ADHLSC or any other cell). In this manner,any treatment or drug regimen that may act synergistically withADHLSC-CM, or an equivalent amount of the protein fraction of ADHLSC-CM,or an equivalent amount of the microvesicles fraction of ADHLSC-CM canbe determined. Remission, survival, prevention, or any other appropriateend-point or marker to be measured in vivo (or selected tissues, organs,or body fluids) for determining a therapeutic effect of the preparationthat is administered to the animals is monitored throughoutpredetermined time periods, taking into account the results obtainedwith available positive or negative control treatments.

A non-exhaustive list of such models and related indications include themodels that are described in the literature cited in the Background, aswell as others such as the D-galactosamine/endotoxin-induced mouse modelof fulminant hepatic failure (FHF), the rat sepsis model of LPS-inducedmulti-organ failure due to LPS injection (generally, in aged rats, LPSis injected and the caecum is perforated, resulting in bacterialperitonitis and all the manifestations of clinical multi-organ failure),the glycerol-induced mouse model of acute renal failure (for example,acute toxic tubular injury in C57Bl/6 mice is induced by intramuscularinjection of 7.5 ml/kg body weight of 50% v/v glycerol solution), theischemia/reperfusion-induced rat model of acute renal failure (forexample, ischemia/reperfusion-type of ARF is induced in anesthetizedrats by timed clamping of both renal pedicles, thereby interrupting theblood supply to the kidneys causing an ischemic insult resulting inacute loss of kidney function, i.e., ARF), the artery ligation-inducedmouse model of ischemia (for example, mice are subjected to right distalfemoral artery ligation using a method known in the art, causingischemia in a hindlimb), the anti-Thy1-1 antibody-induced rat model ofglomerulonephritis (e.g., glomerulonephritis (GN) is induced byintravenous administration at day 0 of 250 μg/100 g weight ofanti-Thy1-1 antibody (Ab) into femoral vein of 6-week-old female Lewisrats), the chemically or cell-based mice models of tumor growth, thecarbon tetrachloride (CCl₄)-induced mouse model of liver fibrosis.

By means of more detailed example, for FHF induction, lethal toxicity oflipopolysaccharide (LPS) on treating SCID (severe combinedimmunodeficiency) mice with D-galactosamine(2-amino-2-deoxy-D-galactose) is developed as previously described(Lehmann V et al., 1987). Amounts of LPS and D-galactosamine areoptimised to obtain 100% lethality within a desired experimental timewindow, such as 8, 16 or 24 hours after injection. By means of moredetailed example, chronic liver disease and liver fibrosis is mimickedin SCID-Beige mice by treatment with carbon tetrachloride (CCl₄) for atleast 3-4 weeks, and potentially for as long as 20 weeks, as previouslydescribed (Perez Tamayo R, 1983). Mice can be treated several times aweek, e.g., three times a week. They can be administered 0.04 cc of a 40percent solution of CCl₄ in olive oil or vehicle by oral gavage. Inmodels such as the above ones, it is observed how survival of theanimals, or other suitable prophylactically or therapeutically relevantend-points or benefits, can be significantly improved by administrationof ADHLSC-CM, or the protein fraction of ADHLSC-CM, or the microvesiclesfraction of ADHLSC-CM when compared to vehicle alone, other types of(non)-conditioned media, and/or other relevant positive or negativecontrols.

On the basis of the results obtained in these models, the actuallypharmaceutically effective amounts of ADHLSC-CM, or the protein fractionof ADHLSC-CM, or the microvesicles fraction of ADHLSC-CM can beappropriately formulated and administered, in particular to humansubjects, by infusion or one or more injections, taking into account thetotal protein concentration and/or the number of cells from which theADHLSC-CM-based preparation has been originally obtained cells isinfused, and then adapting the pharmaceutical composition to thesubject's weight, status, and ongoing treatments. The site ofadministration will depend on the condition or organ treated. Wheresystemic administration is adequate, the composition may be infusedintravenously. Where localised administration is desired, this may beachieved by localised injection. By means of example, delivery to theliver may be performed via the portal vein. Access to the portal vein isby direct puncture under radiological and or ultrasound guidance, via apuncture needle, or via a percutaneous catheter, or via a Port-a-cath Rdevice, or via a Broviac R device inserted surgically in any vesseldraining to the portal vein, preferably the inferior mesenteric vein, ora colonic vein. The catheter can be left in place for several hours,preferably several days, preferably several weeks, or preferably severalmonths up to two years, or preferably longer for repeating infusionswhenever needed.

REFERENCES

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1-13. (canceled)
 14. A method of treating a disorder in a subject inneed thereof, comprising: administering to the subject a therapeuticallyor prophylactically effective amount of a cell-free conditioned medium,wherein the cell-free conditioned medium is produced by culturingadult-derived human liver stem/progenitor cells (ADHLSC) in a cellculture medium and separating the cell culture medium from the cells,wherein the ADHLSC are albumin-positive, vimentin-positive, alpha smoothmuscle actin-positive, cytokeratin-19-negative and CD133-negative.15-16. (canceled)
 17. The method of claim 14, wherein the disorder is afibrotic disorder.
 18. The method of claim 14, wherein the disorder is aliver disorder.
 19. The method of claim 14, wherein the subject issuffering from organ failure, or is in need of organ or celltransplantation.
 20. A method of treating a disorder in a subject inneed thereof, comprising: administering to the subject a therapeuticallyor prophylactically effective amount of a fractioned cell-freecomposition, wherein the fractioned cell-free composition is producedby: culturing adult-derived human liver stem/progenitor cells (ADHLSC)in a cell culture medium and separating the cell culture medium from thecells to produce a cell-free conditioned medium, wherein the ADHLSC arealbumin-positive, vimentin-positive, alpha smooth muscle actin-positive,cytokeratin-19-negative and CD133-negative; and fractioning thecell-free conditioned medium by filtering, enzymatically digesting,centrifuging, adsorbing, and/or separating by chromatography.
 21. Themethod of claim 14, wherein: (a) the cell culture medium is a serum-freemedium; and/or (b) the cell culture medium is separated from ADHLSCafter culturing ADHLSC in the cell culture medium for at least 2 hours,at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours,or at least 24 hours; and/or (c) the adult-derived human liverstem/progenitor cells (ADHLSC) further express at least one markerselected from CD90, CD73, CD44, CD29, alpha-fetoprotein, alpha-1antitrypsin, HNF-4 and MRP2 transporter.
 22. The method of claim 14,wherein the medium comprises hepatocyte growth factor (HGF), vascularendothelial growth factor (VEGF), eotaxin (CCL11), interleukin-6 (IL-6),and interleukin-8 (IL-8) at a concentration of at least 1 ng/mlcomprising the steps of culturing adult-derived human liverstem/progenitor cells (ADHLSC) in a cell culture medium and separatingthe cell culture medium from the cells.
 23. The method of claim 14,wherein the cell-free conditioned medium further comprisesmicrovesicles.
 24. The method of claim 23, wherein the microvesicles aresmaller than 0.40 m.
 25. The method of claim 14, wherein the cell-freeconditioned medium is concentrated at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, or at leastabout 100-fold.
 26. The method of claim 14, wherein the cell-freeconditioned medium is diluted at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, or at leastabout 100-fold.
 27. The method of claim 14, wherein the cell-freeconditioned medium inhibits cell proliferation of hepatic stellate cells(HSCs).
 28. The method of claim 14, wherein the cell-free conditionedmedium inhibits plating efficiency of HSCs.
 29. The method of claim 20,wherein a microvesicle fraction is obtained.
 30. A method of treating adisorder in a subject in need thereof, comprising: administering to thesubject a therapeutically or prophylactically effective amount of amicrovesicle fraction of a cell-free composition, wherein themicrovesicle fraction of the cell-free composition is produced by:culturing adult-derived human liver stem/progenitor cells (ADHLSC) in acell culture medium and separating the cell culture medium from thecells to produce a cell-free conditioned medium, wherein the ADHLSC arealbumin-positive, vimentin-positive, alpha smooth muscle actin-positive,cytokeratin-19-negative and CD133-negative; fractioning the cell-freeconditioned medium by filtering, enzymatically digesting, centrifuging,adsorbing, and/or separating by chromatography; and obtaining amicrovesicle fraction of the cell-free composition.